Polymer-encapsulated reverse micelles

ABSTRACT

A method for encapsulating nucleic acids, particularly siRNAs, shRNAs, microRNAs, gene therapy plasmids, and other oligonucleotides in biodegradable polymers is disclosed, whereby the nucleic acids are formulated into reverse micelles composed of non-toxic and/or naturally-occurring lipids prior to nanoparticle formation by nanoprecipitation. This method can be coupled to other techniques that improve intracellular drug targeting, ultimately enhancing intracellular delivery of the aforementioned nucleic acids.

RELATED APPLICATIONS

This application is a continuation of Application No. PCT/US2008/059483filed Apr. 4, 2008, which claims priority to U.S. ProvisionalApplication No. 60/910,062, filed Apr. 4, 2007, titled “Reverse MicelleNanoprecipitation: A Method for Encapsulating Nucleic Acids inBiodegradable Polymers,” which is incorporated herein by reference inits entirety. Additionally, the contents of any patents, patentapplications, and references cited throughout this specification arehereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number U54CA119349 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF INVENTION

The present invention generally relates to pharmaceutical compositionscomprising reverse micelle particles useful in the treatment ofdiseases.

BACKGROUND

The delivery of a drug to a patient with controlled-release of theactive ingredient has been an active area of research for decades andhas been fueled by the many recent developments in polymer science. Inaddition, controlled release polymer systems can be designed to providea drug level in the optimum range over a longer period of time thanother drug delivery methods, thus increasing the efficacy of the drugand minimizing problems with patient compliance.

Biodegradable particles have been developed as sustained releasevehicles used in the administration of small molecule drugs, proteins,peptide drugs, and nucleic acids. The drugs are typically encapsulatedin a polymer matrix which is biodegradable and biocompatible. As thepolymer is degraded and/or as the drug diffuses out of the polymer, thedrug is released into the body.

Currently, many methods exist for forming nanoparticles of biodegradablepolymers. However, due to the slow partitioning of hydrophilic moleculesinto the hydrophobic nanoparticle core, methods of encapsulating chargedhydrophilic agents, such as nucleic acids, have been plagued by lowencapsulation yield, low drug-to-carrier weight ratios,irreproducibility, and often require emulsification at high shear ratesto achieve nanoparticle size, resulting in product losses.

Thus, there is a need for a method of incorporating hydrophilictherapeutic agents into nanoparticles.

SUMMARY OF THE INVENTION

The present invention provides a method for efficiently encapsulatingtherapeutic agents, e.g., nucleic acids of potential therapeutic ordiagnostic interest, in biodegradable polymers using a nanoprecipitationmethod by first forming non-toxic reverse micelles. These reversemicelles effectively entrap therapeutic agents, such as hydrophilictherapeutic agents, and disperse them into a nanosuspension,facilitating their incorporation into the hydrophobic core ofbiodegradable polymers. This technique improves: encapsulation yield,drug-to-carrier weight ratio, and reproducibility of nanoparticleformation, without requiring the use of potentially damagingemulsification techniques. Further, the methods of the disclosedinvention use components, or segments of components, that arenaturally-derived or FDA-approved for use in humans, thereby generatingclinically relevant drug delivery vehicles.

As such, the present invention is directed toward a pharmaceuticalcomposition, comprising a plurality of target-specific stealthnanoparticles comprising a reverse micelle, a polymeric matrix, atargeting moiety, and a therapeutic agent. In one embodiment, thenanoparticle has an amount of targeting moiety effective for thetreatment of cancer in a subject in need thereof. In another embodiment,the targeting moiety is an aptamer.

In one embodiment, the polymeric matrix of the reverse micellenanoparticle comprises two or more polymers, such as polyethylenes,polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, or polyamines, orcombinations thereof. In one embodiment, the polymeric matrix comprisesone or more polyesters, polyanhydrides, polyethers, polyurethanes,polymethacrylates, polyacrylates or polycyanoacrylates. In a particularembodiment, the polymeric matrix comprises a polyalkylene glycol, suchas polyethylene glycol. In another embodiment, the polymeric matrixcomprises PLGA, PLA, PGA, or a polycaprolactone. In still anotherembodiment, the polymeric matrix comprises a copolymer of two or morepolymers, such as a copolymer of PLGA or PLA and PEG. The polymericmatrix can comprise PLGA or PLA and a copolymer of PLGA or PLA and PEG.

In another embodiment, the polymeric matrix comprises a lipid-terminatedpolyalkylene glycol and a polyester, such as a lipid-terminated PEG andPLGA. As described below, the lipid can be of the Formula V, such as 1,2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts thereof.

In one embodiment of the reverse micelle nanoparticle of the invention,a portion of the polymer matrix is covalently bound to the targetingmoiety, such as via the free terminus of PEG, or via a carboxyl group atthe free terminus of PEG, or via a maleimide functional group at thefree terminus of PEG.

In one embodiment, the reverse micelle nanoparticle of the invention hasa ratio of ligand-bound polymer to non-functionalized polymer effectivefor the treatment of cancer.

The therapeutic agent can be encapsulated within the reverse micelle ofthe nanoparticle. The therapeutic agent can be a nucleic acid, such asnatural or unnatural siRNAs, shRNAs, microRNAs, ribozymes, DNA plasmids,aptamers, antisense oligonucleotides, randomized oligonucleotides, orribozymes. In a particular embodiment, the therapeutic agent is ansiRNA, such as an siRNA molecule that is complementary to tumor-relatedtargets, such as an anti-PLK1 siRNA or an anti-VEGF siRNA.

In one embodiment of the nanoparticle of the invention, the reversemicelle comprises an amphipathic lipid, such as lecithin,phosphatidylcholine, lipid A, cholesterol, dolichol, shingosine,sphingomyelin, ceramide, cerebroside, sulfatide, glycosylceramide,phytosphingosine, phosphatidylethanolamine, phosphatidylglycerol,phosphatidylinositol, phosphatidylserine, cardiolipin, phophatidic acid,and lysophophatides. In one embodiment, the therapeutic agent is anucleic acid, and the ratio of amphipathic lipid to nucleic acid isapproximately 33:1.

In another aspect, the invention provides a method of treating prostatecancer in a subject in need thereof, comprising administering to thesubject an effective amount of the pharmaceutical composition of theinvention. The pharmaceutical composition can be administered directlyto the prostate of a subject or directly to prostate cancer cells, suchas administered directly to prostate cancer cells by injection intotissue comprising the prostate cancer cells. In another embodiment, thepharmaceutical composition is administered to the subject byimplantation of nanoparticles at or near prostate cancer cells duringsurgical removal of a tumor. The pharmaceutical composition can beadministered systemically, such as intravenously.

The pharmaceutical composition of the invention can be used in themanufacture of a medicament for the treatment of cancer, such asprostate cancer. The pharmaceutical composition can be administeredintravenously.

In another embodiment, the nanoparticle has an amount of targetingmoiety effective for the treatment of a cancer wherein PSMA is expressedon the surface of cancer cells or in the tumor neovasculature in asubject in need thereof. The PSMA-related indication can be selectedfrom the group consisting of prostate cancer, non-small cell lungcancer, colorectal carcinoma, and glioblastoma.

In another aspect, the invention provides a nanoparticle comprising areverse micelle, wherein the reverse micelle is encapsulated by apolymer, and a therapeutic agent, wherein the therapeutic agent is anucleic acid.

In another aspect, the invention provides a particle for drug deliverycomprising a hydrophilic agent entrapped in a reverse micelle, whereinthe interior of the reverse micelle is hydrophilic and the exterior ishydrophobic, and wherein the reverse micelle is encapsulated by apolymer. The hydrophilic agent can be a nucleic acid, such as a DNA, anRNA, an siRNA, shRNA, microRNA, aptamer, plasmid, chromosome, ribozymes,or antisense oligonucleotide, a protein, a small molecule, or a chargedagent.

The reverse micelle of the particle of the invention can comprise anamphipathic lipid, such as lecithin, DPAP, phosphatidylcholine, lipid A,cholesterol, dolichol, shingosine, sphingomyelin, ceramide, cerebroside,sulfatide, glycosylceramide, phytosphingosine, phosphatidylethanolamine,phosphatidylglycerol, phosphatidylinositol, phosphatidylserine,cardiolipin, phophatidic acid, and lysophophatides. In one embodiment,the amphipathic lipid is neutral. In another embodiment, the amphipathiclipid is cationic.

In another embodiment of the particle, the polymer is poly(lactic acid),poly(glycolic acid), poly(lactic-co-glycolic acid), poly(caprolactone),or poly (anhydride). The overall charge on the particle can be negativeor neutral.

Furthermore, the particle of the invention can be combined with apharmaceutically acceptable excipient to form a pharmaceuticalcomposition.

In another aspect, the invention provides a method of preparing aparticle of the invention, the method comprising steps of: providing atherapeutic agent; dissolving the therapeutic agent with an amphipathiclipid in a volatile, water-miscible organic solvent; forming reversemicelles, wherein the interior of the reverse micelle is hydrophilic andcontains the therapeutic agent, and the exterior of the reverse micelleis hydrophobic; adding a polymer to the mixture of reverse micelles;combining the resulting mixture with a hydrophilic non-solvent that thepolymer is not soluble in to form nanoparticle by rapid diffusion of thesolvent into the non-solvent, and evaporation of the solvent. Thetherapeutic agent used in the method can be a hydrophilic agent, such asa nucleic acid. The solvent can be tetrahydrofuran, acetone,acetonitrile, or dimethylformamide. The non-solvent is water, ethanol,methanol, or mixtures thereof. The polymer of the method comprises acopolymer of two or more polymers, such as a copolymer of PLGA and PEG,or PLA and PEG.

In one embodiment, the nanoparticles of the invention is less than 100nm in diameter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the effect of amphipathic lipid concentration onencapsulation particle size.

FIG. 2 demonstrates the effect of amphipathic lipid concentration onparticle zeta potential.

FIG. 3 demonstrates the effect of amphipathic lipid concentration onparticle encapsulation efficiency.

FIG. 4 demonstrates the effect of amphipathic lipid concentration onweight percent nucleic acids in the nanoparticles that are formed usingthe methods of the invention.

FIG. 5 demonstrates the effect of amphipathic lipid concentration onparticle encapsulation efficiency.

FIG. 6 demonstrates the effect of amphipathic lipid concentration ontherapeutic agent release over time.

FIG. 7 demonstrates cell uptake of the polymer-encapsulated reversemicelles of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to particles, and, inparticular, polymer-encapsulated reverse micelles. One aspect of theinvention is directed to a method of developing polymer-encapsulatedreverse micelles with desired properties, wherein the micelles contain atargeting moiety (also referred to as a “ligand”), such as an aptamer.One or more of the polymers may be a biocompatible polymer (e.g.,homopolymer, copolymer or block copolymer), wherein the biocompatiblepolymer may be conjugated to a targeting moiety such as an aptamer. Insome cases, the polymer-encapsulated reverse micelles may contain atherapeutic agent, e.g., a drug.

In one embodiment, the polymer-encapsulated reverse micelle of theinvention has an amount of targeting moiety (e.g., an aptamer) effectivefor the treatment of a disease, e.g., prostate cancer, in a subject inneed thereof. In certain embodiments, the targeting moiety is conjugatedto a polymer. Thus, in one embodiment, the polymer-encapsulated reversemicelle comprises a polymer that is functionalized with a targetingmoiety. In other embodiments, the polymer-encapsulated reverse micellecomprises a certain ratio of targeting moiety-conjugated polymer tonon-functionalized polymer. The reverse micelle can have an optimizedratio of these two polymers, such that an effective amount of ligand isassociated with the reverse micelle for treatment of a disease, e.g.,prostate cancer. For example, increased ligand density (e.g., on aPLGA-PEG copolymer) will increase target binding (cell binding/targetuptake), making the reverse micelle “target specific.” Alternatively, acertain concentration of non-functionalized polymer (e.g.,non-functionalized PLGA-PEG copolymer) in the reverse micelle cancontrol inflammation and/or immunogenicity (i.e., the ability to provokean immune response), and allow the reverse micelle to have a circulationhalf-life that is adequate for the treatment of cancer (e.g., prostatecancer). Furthermore, the non-functionalized polymer can lower the rateof clearance from the circulatory system via the reticuloendothelialsystem. Thus, the non-functionalized polymer gives the reverse micelle“stealth” characteristics. In a particular embodiment, the stealthpolymer is PEG. Additionally, the non-functionalized polymer balances anotherwise high concentration of ligands, which can otherwise accelerateclearance by the subject, resulting in less delivery to the targetcells.

By having targeting moieties, the “target specific” reverse micelles areable to efficiently bind to or otherwise associate with a biologicalentity, for example, a membrane component or cell surface receptor.Targeting of a therapeutic agent (e.g., to a particular tissue or celltype, to a specific diseased tissue but not to normal tissue, etc.) isdesirable for the treatment of tissue specific diseases such as cancer(e.g. prostate cancer). For example, in contrast to systemic delivery ofa cytotoxic anti-cancer agent, targeted delivery could prevent the agentfrom killing healthy cells. Additionally, targeted delivery can allowfor the administration of a lower dose of the agent, which could reducethe undesirable side effects commonly associated with traditionalchemotherapy. As discussed herein, the target specificity of the reversemicelle of the invention will be maximized by optimizing the liganddensity on the reverse micelle.

Polymer-Encapsulated Reverse Micelles

For the purposes of the invention, the term “reverse micelle” refereesto a micelle in which a hydrophilic component is in the inner portion ofthe micelle. A typical micelle in aqueous solution forms an aggregatewith the hydrophilic head regions in contact with surrounding solution,sequestering the hydrophobic tail regions in the micelle center. Thus,the hydrophilic portion of the polymer-encapsulated reverse micelle,which is on the inner portion of the reverse micelle, can effectivelyentrap therapeutic agents, including hydrophilic therapeutic agents suchas nucleic acids.

The reverse micelle of the present invention comprises an amphipathiclipid. As used herein, the term “amphipathic” refers to a property wherea molecule has both a polar portion and a non-polar portion. Often, anamphipathic compound has a polar head attached to a long hydrophobictail. In some embodiments, the polar portion is soluble in water, whilethe non-polar portion is insoluble in water. In addition, the polarportion may have either a formal positive charge, or a formal negativecharge. Alternatively, the polar portion may have both a formal positiveand a negative charge, and be a zwitterion or inner salt. For purposesof the invention, the amphipathic compound can be, but is not limitedto, one or a plurality of the following: naturally derived lipids,surfactants, or synthesized compounds with both hydrophilic andhydrophobic moieties.

Specific examples of amphipathic compounds include, but are not limitedto, lecithin, phosphatidylcholine, lipid A, cholesterol, dolichol,shingosine, sphingomyelin, ceramide, cerebroside, sulfatide,glycosylceramide, phytosphingosine, phosphatidylethanolamine,phosphatidylglycerol, phosphatidylinositol, phosphatidylserine,cardiolipin, phophatidic acid, and lysophophatides.

In one embodiment, the amphipathic lipid is dimethyldiotadecylammoniumbromide (DDAB).

In a particular embodiment, an amphipathic component that can be used toform the reverse micelle is lecithin, and, in particular,phosphatidylcholine. Lecithin is an amphiphilic lipid and, as such,forms a phospholipid bilayer having the hydrophilic (polar) heads facingtheir surroundings, which are oftentimes aqueous, and the hydrophobictails facing each other. Lecithin has an advantage of being a naturallipid that is available from, e.g., soybean, and already has FDAapproval for use in other delivery devices. In addition, a mixture oflipids such as lethicin is more advantageous than one single pure lipid.

In certain embodiments of the invention, the amphiphilic layer of thenanoparticle, e.g., the layer of lecithin, is a monolayer, meaning thelayer is not a phospholipid bilayer, but exists as a single continuousor discontinuous layer around, or within, the nanoparticle. Theamphiphilic layer is “associated with” the nanoparticle of theinvention, meaning it is positioned in some proximity to the polymericmatrix, such as surrounding the outside of the polymeric shell, ordispersed within the polymers that make up the nanoparticle.

In one embodiment, the ratio of amphipathic lipid to nucleic acid isapproximately 50:1, 40:1, 30:1, 20:1, and 10:1, molar. In a particularembodiment, the ratio of amphipathic lipid to nucleic acid isapproximately 33:1, molar.

In some embodiments, a therapeutic agent and/or targeting moiety (e.g.,an aptamer) can be associated with the polymer-encapsulated reversemicelle. In some embodiments, the targeting moiety can be covalentlyassociated with the surface of the polymer that encapsulates the reversemicelle. In some embodiments, covalent association is mediated by alinker. In some embodiments, the therapeutic agent can be encapsulatedwithin the reverse micelle.

Thus, in one embodiment, the invention provides a polymer-encapsulatedreverse micelle comprising 1) PLGA; 2) PEG; 3) an amphipathic lipid(e.g., lecithin); and 4) a covalently attached targeting moiety. In oneembodiment, the PLGA and PEG are copolymers, and the targeting moiety iscovalently bound to PEG. In another embodiment, the PEG is bound toDSPE, which self assembles with PLGA, and the low molecular weight PSMAligand is covalently bound to PEG.

In another embodiment, the invention comprises a polymer-encapsulatedreverse micelle comprising 1) a polymeric matrix comprising abiodegradable polymer; 2) an amphipathic lipid; 3) a stealth polymer,and 4) a covalently attached targeting moiety. In another embodiment,the invention comprises a polymer-encapsulated reverse micellecomprising 1) a polymeric matrix comprising a biodegradable polymer; 2)lecithin; 3) a stealth polymer, and 4) a covalently attached targetingmoiety.

Any polymer may be used in accordance with the present invention.Polymers may be natural or unnatural (synthetic) polymers. Polymers maybe homopolymers or copolymers comprising two or more monomers. In termsof sequence, copolymers may be random, block, or comprise a combinationof random and block sequences. Typically, polymers in accordance withthe present invention are organic polymers.

A “polymer,” as used herein, is given its ordinary meaning as used inthe art, i.e., a molecular structure comprising one or more repeat units(monomers), connected by covalent bonds. The repeat units may all beidentical, or in some cases, there may be more than one type of repeatunit present within the polymer. In some cases, the polymer isbiologically derived, i.e., a biopolymer. Non-limiting examples ofpolymers include peptides or proteins (i.e., polymers of various aminoacids), or nucleic acids such as DNA or RNA. In some cases, additionalmoieties may also be present in the polymer, for example biologicalmoieties such as those described below.

If more than one type of repeat unit is present within the polymer, thenthe polymer is said to be a “copolymer.” It is to be understood that inany embodiment employing a polymer, the polymer being employed may be acopolymer in some cases. The repeat units forming the copolymer may bearranged in any fashion. For example, the repeat units may be arrangedin a random order, in an alternating order, or as a “block” copolymer,i.e., comprising one or more regions each comprising a first repeat unit(e.g., a first block), and one or more regions each comprising a secondrepeat unit (e.g., a second block), etc. Block copolymers may have two(a diblock copolymer), three (a triblock copolymer), or more numbers ofdistinct blocks.

It should be understood that, although the terms “first,” “second,” etc.may be used herein to describe various elements, including polymericcomponents, these terms should not be construed as being limiting (e.g.,describing a particular order or number of elements), but rather, asbeing merely descriptive, i.e., labels that distinguish one element fromanother, as is commonly used within the field of patent law. Thus, forexample, although one embodiment of the invention may be described ashaving a “first” element present and a “second” element present, otherembodiments of the invention may have a “first” element present but no“second” element present, a “second” element present but no “first”element present, two (or more) “first” elements present, and/or two (ormore) “second” elements present, etc., and/or additional elements suchas a “first” element, a “second” element, and a “third” element, withoutdeparting from the scope of the present invention.

Various embodiments of the present invention are directed to copolymers,which, in particular embodiments, describes two or more polymers (suchas those described herein) that have been associated with each other,usually by covalent bonding of the two or more polymers together. Thus,a copolymer may comprise a first polymer and a second polymer, whichhave been conjugated together to form a block copolymer where the firstpolymer is a first block of the block copolymer and the second polymeris a second block of the block copolymer. Of course, those of ordinaryskill in the art will understand that a block copolymer may, in somecases, contain multiple blocks of polymer, and that a “block copolymer,”as used herein, is not limited to only block copolymers having only asingle first block and a single second block. For instance, a blockcopolymer may comprise a first block comprising a first polymer, asecond block comprising a second polymer, and a third block comprising athird polymer or the first polymer, etc. In some cases, block copolymerscan contain any number of first blocks of a first polymer and secondblocks of a second polymer (and in certain cases, third blocks, fourthblocks, etc.). In addition, it should be noted that block copolymers canalso be formed, in some instances, from other block copolymers.

For example, a first block copolymer may be conjugated to anotherpolymer (which may be a homopolymer, a biopolymer, another blockcopolymer, etc.), to form a new block copolymer containing multipletypes of blocks, and/or to other moieties (e.g., to non-polymericmoieties).

In some embodiments, the polymer (e.g., copolymer, e.g., blockcopolymer) is amphiphilic, i.e., having a hydrophilic portion and ahydrophobic portion, or a relatively hydrophilic portion and arelatively hydrophobic portion. A hydrophilic polymer is one generallythat attracts water and a hydrophobic polymer is one that generallyrepels water. A hydrophilic or a hydrophobic polymer can be identified,for example, by preparing a sample of the polymer and measuring itscontact angle with water (typically, the polymer will have a contactangle of less than 60°, while a hydrophobic polymer will have a contactangle of greater than about) 60°. In some cases, the hydrophilicity oftwo or more polymers may be measured relative to each other, i.e., afirst polymer may be more hydrophilic than a second polymer. Forinstance, the first polymer may have a smaller contact angle than thesecond polymer.

In one set of embodiments, a polymer (e.g., copolymer, e.g., blockcopolymer) of the present invention includes a biocompatible polymer,i.e., the polymer that does not typically induce an adverse responsewhen inserted or injected into a living subject, for example, withoutsignificant inflammation and/or acute rejection of the polymer by theimmune system, for instance, via a T-cell response. It will berecognized, of course, that “biocompatibility” is a relative term, andsome degree of immune response is to be expected even for polymers thatare highly compatible with living tissue. However, as used herein,“biocompatibility” refers to the acute rejection of material by at leasta portion of the immune system, i.e., a non-biocompatible materialimplanted into a subject provokes an immune response in the subject thatis severe enough such that the rejection of the material by the immunesystem cannot be adequately controlled, and often is of a degree suchthat the material must be removed from the subject. One simple test todetermine biocompatibility is to expose a polymer to cells in vitro;biocompatible polymers are polymers that typically will not result insignificant cell death at moderate concentrations, e.g., atconcentrations of 50 micrograms/10⁶ cells. For instance, a biocompatiblepolymer may cause less than about 20% cell death when exposed to cellssuch as fibroblasts or epithelial cells, even if phagocytosed orotherwise uptaken by such cells. Non-limiting examples of biocompatiblepolymers that may be useful in various embodiments of the presentinvention include polydioxanone (PDO), polyhydroxyalkanoate,polyhydroxybutyrate, poly(glycerol sebacate), polyglycolide,polylactide, PLGA, polycaprolactone, or copolymers or derivativesincluding these and/or other polymers.

In certain embodiments, the biocompatible polymer is biodegradable,i.e., the polymer is able to degrade, chemically and/or biologically,within a physiological environment, such as within the body. Forinstance, the polymer may be one that hydrolyzes spontaneously uponexposure to water (e.g., within a subject), the polymer may degrade uponexposure to heat (e.g., at temperatures of about 37° C.). Degradation ofa polymer may occur at varying rates, depending on the polymer orcopolymer used. For example, the half-life of the polymer (the time atwhich 50% of the polymer is degraded into monomers and/or othernonpolymeric moieties) may be on the order of days, weeks, months, oryears, depending on the polymer. The polymers may be biologicallydegraded, e.g., by enzymatic activity or cellular machinery, in somecases, for example, through exposure to a lysozyme (e.g., havingrelatively low pH). In some cases, the polymers may be broken down intomonomers and/or other nonpolymeric moieties that cells can either reuseor dispose of without significant toxic effect on the cells (forexample, polylactide may be hydrolyzed to form lactic acid,polyglycolide may be hydrolyzed to form glycolic acid, etc.).

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEGylated polymers andcopolymers of lactide and glycolide (e.g., PEGylated PLA, PEGylated PGA,PEGylated PLGA, and derivatives thereof. In some embodiments, polyestersinclude, for example, polyanhydrides, poly(ortho ester) PEGylatedpoly(ortho ester), poly(caprolactone), PEGylated poly(caprolactone),polylysine, PEGylated polylysine, poly(ethylene inline), PEGylatedpoly(ethylene imine), poly(L-lactide-co-L-lysine), poly(serine ester),poly(4-hydroxy-L-proline ester), poly[a-(4-aminobutyl)-L-glycolic acid],and derivatives thereof.

In some embodiments, the polymer may be PLGA. PLGA is a biocompatibleand biodegradable co-polymer of lactic acid and glycolic acid, andvarious forms of PLGA are characterized by the ratio of lacticacid:glycolic acid. Lactic acid can be L-lactic acid, D-lactic acid, orD,L-lactic acid. The degradation rate of PLGA can be adjusted byaltering the lactic acid-glycolic acid ratio. In some embodiments, PLGAto be used in accordance with the present invention is characterized bya lactic acid:glycolic acid ratio of approximately 85:15, approximately75:25, approximately 60:40, approximately 50:50, approximately 40:60,approximately 25:75, or approximately 15:85.

In particular embodiments, by optimizing the ratio of lactic acid toglycolic acid monomers in the polymer of the reverse micelle (e.g., thePLGA block copolymer or PLGA-PEG block copolymer), reverse micelleparameters such as water uptake, therapeutic agent release (e.g.,“controlled release”) and polymer degradation kinetics can be optimized.

In some embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid polyacrylamide, aminoalkyl methacrylate copolymer,glycidyl methacrylate copolymers, polycyanoacrylates, and combinationscomprising one or more of the foregoing polymers. The acrylic polymermay comprise fully-polymerized copolymers of acrylic and methacrylicacid esters with a low content of quaternary ammonium groups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids (e.g. DNA, RNA, or derivatives thereof).Amine-containing polymers such as poly(lysine) (Zauner et al., 1998,Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, BioconjugateChem., 6:7), poly(ethylene imine) (PEI; Boussif et al, 1995, Proc. Natl.Acad. Sci., USA, 1995, 92:7297), and poly(amidoamine) dendrimers(Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897;Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993,Bioconjugate Chem., 4:372) are positively-charged at physiological pH,form ion pairs with nucleic acids, and mediate transfection in a varietyof cell lines.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains (Putnam et al., 1999, Macromolecules, 32:3658;Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Urn et al., 1999, J.Am. Chem. Soc., 121:5633; and Zhou et al, 1990, Macromolecules,23:3399). Examples of these polyesters includepoly(L-lactide-co-L-lysine) (Barrera et al, 1993, J. Am. Chem. Soc.,115:11010), poly(serine ester) (Zhou et al, 1990, Macromolecules,23:3399), poly(4-hydroxy-L-proline ester) (Putnam et al, 1999,Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc.,121:5633). Poly(4-hydroxy-L-proline ester) was demonstrated to condenseplasmid DNA through electrostatic interactions, and to mediate genetransfer (Putnam et al., 1999, Macromolecules, 32:3658; and Lim et al.,1999, J. Am. Chem. Soc., 121:5633). These new polymers are less toxicthan poly(lysine) and PEI, and they degrade into non-toxic metabolites.

A polymer (e.g., copolymer, e.g., block copolymer) containingpoly(ethylene glycol) repeat units is also referred to as a “PEGylated”polymer. Such polymers can control inflammation and/or immunogenicity(i.e., the ability to provoke an immune response) and/or lower the rateof clearance from the circulatory system via the reticuloendothelialsystem, due to the presence of the poly(ethylene glycol) groups.

PEGylation may also be used, in some cases, to decrease chargeinteraction between a polymer and a biological moiety, e.g., by creatinga hydrophilic layer on the surface of the polymer, which may shield thepolymer from interacting with the biological moiety. In some cases, theaddition of poly(ethylene glycol) repeat units may increase plasmahalf-life of the polymer (e.g., copolymer, e.g., block copolymer), forinstance, by decreasing the uptake of the polymer by the phagocyticsystem while decreasing transfection/uptake efficiency by cells. Thoseof ordinary skill in the art will know of methods and techniques forPEGylating a polymer, for example, by using EDC(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) andNHS(N-hydroxysuccinimide) to react a polymer to a PEG group terminatingin an amine, by ring opening polymerization techniques (ROMP), or thelike.

In addition, certain embodiments of the invention are directed towardscopolymers containing poly(ester-ether)s, e.g., polymers having repeatunits joined by ester bonds (e.g., R—C(O)—O—R′ bonds) and ether bonds(e.g., R—O—R′ bonds). In some embodiments of the invention, abiodegradable polymer, such as a hydrolyzable polymer, containingcarboxylic acid groups, may be conjugated with poly(ethylene glycol)repeat units to form a poly(ester-ether).

In a particular embodiment, the molecular weight of the polymers of thereverse micelles of the invention are optimized for effective treatmentof cancer, e.g., prostate cancer. For example, the molecular weight ofthe polymer influences reverse micelle particle degradation rate(particularly when the molecular weight of a biodegradable polymer isadjusted), solubility, water uptake, and drug release kinetics (e.g.“controlled release”). As a further example, the molecular weight of thepolymer can be adjusted such that the reverse micelle biodegrades in thesubject being treated within a reasonable period of time (ranging from afew hours to 1-2 weeks, 3-4 weeks, 5-6 weeks, 7-8 weeks, etc.). Inparticular embodiments of a reverse micelle comprising a copolymer ofPEG and PLGA, the PEG has a molecular weight of 1,000-20,000, e.g.,5,000-20,000, e.g., 10,000-20,000, and the PLGA has a molecular weightof 5,000-100,000, e.g., 20,000-70,000, e.g., 20,000-50,000.

In certain embodiments, the polymers of the reverse micelles may beconjugated to a lipid. The polymer may be, for example, alipid-terminated PEG. As described below, the lipid portion of thepolymer can be used for self assembly with another polymer, facilitatingthe formation of a reverse micelle. For example, a hydrophilic polymercould be conjugated to a lipid that will self assemble with ahydrophobic polymer.

In some embodiments, lipids are oils. In general, any oil known in theart can be conjugated to the polymers used in the invention. In someembodiments, an oil may comprise one or more fatty acid groups or saltsthereof. In some embodiments, a fatty acid group may comprisedigestible, long chain (e.g., C₈-C₅₀), substituted or unsubstitutedhydrocarbons. In some embodiments, a fatty acid group may be a C₁₀-C₂₀fatty acid or salt thereof. In some embodiments, a fatty acid group maybe a C₁₅-C₂₀ fatty acid or salt thereof. In some embodiments, a fattyacid may be unsaturated. In some embodiments, a fatty acid group may bemonounsaturated. In some embodiments, a fatty acid group may bepolyunsaturated. In some embodiments, a double bond of an unsaturatedfatty acid group may be in the cis conformation. In some embodiments, adouble bond of an unsaturated fatty acid may be in the transconformation.

In some embodiments, a fatty acid group may be one or more of butyric,caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linolenic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In a particular embodiment, the lipid is of the Formula V:

and salts thereof, wherein each R is, independently, C₁₋₃₀ alkyl. In oneembodiment of Formula V, the lipid is 1,2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts thereof,e.g., the sodium salt.

The properties of the polymers discussed herein, and methods forpreparing them, are well known in the art (see, for example, U.S. Pat.Nos. 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148;5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665;5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al,2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am. Chem. Soc.,123:2460; Langer, 2000, Ace. Chem. Res., 33:94; Langer, 1999, J.Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev., 99:3181).More generally, a variety of methods for synthesizing suitable polymersare described in Concise Encyclopedia of Polymer Science and PolymericAmines and Ammonium Salts, Ed. by Goethals, Pergamon Press, 1980;Principles of Polymerization by Odian, John Wiley & Sons, FourthEdition, 2004; Contemporary Polymer Chemistry by Allcock et al.,Prentice-Hall, 1981; Deming et al, 1997, Nature, 390:386; and in U.S.Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.

In still another set of embodiments, a particle (comprising, e.g., acopolymer, e.g., a block copolymer) of the present invention includes atherapeutic agent, i.e., an agent that has a therapeutic or prophylacticeffect when given to a subject. Examples of therapeutic moieties to beused with the reverse micelles of the present invention includetherapeutic nucleic acids, antineoplastic or cytostattc agents or otheragents with anticancer properties, or a combination thereof.

In some cases, the polymer-encapsulated reverse micelle has acharacteristic dimension of less than about 1 micrometer. For example,the particle may have a characteristic dimension of the particle may beless than about 300 nm, less than about 200 nm, less than about 150 nm,less than about 100 nm, less than about 50 nm, less than about 30 nm,less than about 10 nm, less than about 3 nm, or less than about 1 nm insome cases. In particular embodiments, the nanoparticle of the presentinvention has a diameter of less than 100 nm.

Yet another aspect of the invention is directed to reverse micelleshaving more than one polymer present, and libraries involving suchreverse micelles. For example, in one set of embodiments, reversemicelles may contain more than one distinguishable polymers (e.g.,copolymers, e.g., block copolymers), and the ratios of the two (or more)polymers may be independently controlled, which allows for the controlof properties of the particle. For instance, a first polymer may be apolymeric conjugate comprising a targeting moiety and a biocompatibleportion, and a second polymer may comprise a biocompatible portion butnot contain the targeting moiety. Control of the amounts of thesepolymers within the reverse micelle may thus be used to control variousphysical, biological, or chemical properties of the reverse micelle, forinstance, the size of the reverse micelle (e.g., by varying themolecular weights of one or both polymers), the surface charge (e.g., bycontrolling the ratios of the polymers if the polymers have differentcharges or terminal groups), the surface hydrophilicity (e.g., if thepolymers have different molecular weights and/or hydrophilicities), thesurface density of the targeting moiety (e.g., by controlling the ratiosof the two or more polymers), etc.

As a specific example, a reverse micelle may comprise a first polymercomprising a poly(ethylene glycol) and a targeting moiety conjugated tothe poly(ethylene glycol), and a second polymer comprising thepoly(ethylene glycol) but not the targeting moiety, or comprising boththe poly(ethylene glycol) and the targeting moiety, where thepoly(ethylene glycol) of the second polymer has a different length (ornumber of repeat units) than the poly(ethylene glycol) of the firstpolymer. As another example, a reverse micelle may comprise a firstpolymer comprising a first biocompatible portion and a targeting moiety,and a second polymer comprising a second biocompatible portion differentfrom the first biocompatible portion (e.g., having a differentcomposition, a substantially different number of repeat units, etc.) andthe targeting moiety. As yet another example, a first polymer maycomprise a biocompatible portion and a first targeting moiety, and asecond polymer may comprise a biocompatible portion and a secondtargeting moiety different from the first targeting moiety.

Targeting Moieties

In yet another set of embodiments a reverse micelle of the presentinvention includes a targeting moiety, i.e., a moiety able to bind to orotherwise associate with a biological entity, for example, a membranecomponent, a cell surface receptor, prostate specific membrane antigen,or the like. For example, a targeting portion may cause the particles tobecome localized to a tumor, a disease site, a tissue, an organ, a typeof cell, etc. within the body of a subject, depending on the targetingmoiety used. The term “bind” or “binding,” as used herein, refers to theinteraction between a corresponding pair of molecules or portionsthereof that exhibit mutual affinity or binding capacity, typically dueto specific or non-specific binding or interaction, including, but notlimited to, biochemical, physiological, and/or chemical interactions.“Biological binding” defines a type of interaction that occurs betweenpairs of molecules including proteins, nucleic acids, glycoproteins,carbohydrates, hormones, or the like. The term “binding partner” refersto a molecule that can undergo binding with a particular molecule.“Specific binding” refers to molecules, such as polynucleotides, thatare able to bind to or recognize a binding partner (or a limited numberof binding partners) to a substantially higher degree than to other,similar biological entities. In one set of embodiments, the targetingmoiety has an affinity (as measured via a disassociation constant) ofless than about 1 micromolar, at least about 10 micromolar, or at leastabout 100 micromolar.

The targeting moiety (e.g., an aptamer) can be covalently bonded thepolymeric matrix component of the reverse micelle. In some embodiments,the targeting moiety can be covalently associated with the surface ofthe polymeric component (e.g., PEG). In some embodiments, covalentassociation is mediated by a linker. In some embodiments, thetherapeutic agent can be associated with the surface of, encapsulatedwithin, surrounded by, and/or dispersed throughout the polymeric matrix.

A targeting moiety may be a nucleic acid, polypeptide, glycoprotein,carbohydrate, or lipid. For example, a targeting moiety can be a nucleicacid targeting moiety (e.g. an aptamer) that binds to a cell typespecific marker. In general, an aptamer is an oligonucleotide (e.g.,DNA, RNA, or an analog or derivative thereof) that binds to a particulartarget, such as a polypeptide. In some embodiments, a targeting moietymay be a naturally occurring or synthetic ligand for a cell surfacereceptor, e.g., a growth factor, hormone, LDL, transferrin, etc. Atargeting moiety can be an antibody, which term is intended to includeantibody fragments, characteristic portions of antibodies, single chaintargeting moieties can be identified, e.g., using procedures such asphage display. This widely used technique has been used to identify cellspecific ligands for a variety of different cell types.

In some embodiments, targeting moieties bind to an organ, tissue, cell,extracellular matrix component, and/or intracellular compartment that isassociated with a specific developmental stage or a specific diseasestate. In some embodiments, a target is an antigen on the surface of acell, such as a cell surface receptor, an integrin, a transmembraneprotein, an ion channel, and/or a membrane transport protein. In someembodiments, a target is an intracellular protein. In some embodiments,a target is a soluble protein, such as immunoglobulin. In certainspecific embodiments, a target is a tumor marker. In some embodiments, atumor marker is an antigen that is present in a tumor that is notpresent in normal tissue. In some embodiments, a tumor marker is anantigen that is more prevalent in a tumor than in normal tissue. In someembodiments, a tumor marker is an antigen that is more prevalent inmalignant cancer cells than in normal cells.

In some embodiments, a target is preferentially expressed in tumortissues versus normal tissues. For example, when compared withexpression in normal tissues, expression of prostate specific membraneantigen (PSMA) is at least 10-fold overexpressed in malignant prostaterelative to normal tissue, and the level of PSMA expression is furtherup-regulated as the disease progresses into metastatic phases (Silver etal., 1997, Clin. Cancer Res., 3:81).

In some embodiments, inventive targeted reverse micelles comprise lessthan 50% by weight, less than 40% by weight, less than 30% by weight,less than 20% by weight, less than 15% by weight, less than 10% byweight, less than 5% by weight, less than 1% by weight, or less than0.5% by weight of the targeting moiety.

In some embodiments, the targeting moieties are covalently associatedwith the reverse micelle. In some embodiments, covalent association ismediated by a linker.

Nucleic Acid Targeting Moieties

As used herein, a “nucleic acid targeting moiety” is a nucleic acid thatbinds selectively to a target. In some embodiments, a nucleic acidtargeting moiety is a nucleic acid that is associated with a particularorgan, tissue, cell, extracellular matrix component, and/orintracellular compartment. In general, the targeting function of theaptamer is based on the three-dimensional structure of the aptamer. Insome embodiments, binding of an aptamer to a target is typicallymediated by the interaction between the two- and/or three-dimensionalstructures of both the aptamer and the target. In some embodiments,binding of an aptamer to a target is not solely based on the primarysequence of the aptamer, but depends on the three-dimensionalstructure(s) of the aptamer and/or target. In some embodiments, aptamersbind to their targets via complementary Watson-Crick base pairing whichis interrupted by structures (e.g. hairpin loops) that disrupt basepairing.

One of ordinary skill in the art will recognize that any aptamer that iscapable of specifically binding to a target can be used in accordancewith the present invention. In some embodiments, aptamers to be used inaccordance with the present invention may target cancer-associatedtargets. In some embodiments, aptamers to be used in accordance with thepresent invention may target tumor markers.

In certain embodiments, aptamers to be used in accordance with thepresent invention may target prostate cancer associated antigens, suchas PSMA. Exemplary PSMA-targeting aptamers to be used in accordance withthe present invention include, but are not limited to, the A10 aptamer,having a nucleotide sequence of5′-GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCUUGUCAAUCCUCAUCGGCAGACGACUCGCCCGA-3′ (SEQ ID NO: 1) (Lupold et al, 2002, CancerRes, 62:4029), the A9 aptamer, having nucleotide sequence of5′-GGGAGGACGAUGCGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGUUCCCAGACGACUCGCCCGA-3′ (SEQ ID NO: 2) (Lupold et al., 2002, CancerRes., 62:4029; and Chu et al., 2006, Nuc. Acid Res., 34:e73),derivatives thereof, and/or characteristic portions thereof.

In some embodiments, a nucleotide sequence that is homologous to anucleic acid targeting moiety may be used in accordance with the presentinvention. In some embodiments, a nucleotide sequence is considered tobe “homologous” to a nucleic acid targeting moiety if it comprises fewerthan 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 nucleic acid substitutionsrelative to the aptamer. In some embodiments, a nucleotide sequence isconsidered to be “homologous” to a nucleic acid targeting moiety iftheir sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In someembodiments, a nucleic acid sequence is considered to be 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar.Nucleic acids of the present invention (including nucleic acid targetingmoieties and/or functional RNAs to be delivered, e.g., RNAi agents,ribozymes, tRNAs, etc., described in further detail below) may beprepared according to any available technique including, but not limitedto, chemical synthesis, enzymatic synthesis, enzymatic or chemicalcleavage of a longer precursor, etc. Methods of synthesizing RNAs areknown in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotidesynthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.:IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis:methods and applications, Methods in molecular biology, v. 288 (Clifton,N.J.) Totowa, N.J.: Humana Press, 2005).

The nucleic acid that forms the nucleic acid targeting moiety maycomprise naturally occurring nucleosides, modified nucleosides,naturally occurring nucleosides with hydrocarbon linkers (e.g., analkylene) or a polyether linker (e.g., a PEG linker) inserted betweenone or more nucleosides, modified nucleosides with hydrocarbon or PEGlinkers inserted between one or more nucleosides, or a combination ofthereof. In some embodiments, nucleotides or modified nucleotides of thenucleic acid targeting moiety can be replaced with a hydrocarbon linkeror a polyether linker provided that the binding affinity and selectivityof the nucleic acid targeting moiety is not substantially reduced by thesubstitution (e.g., the dissociation constant of the nucleic acidtargeting moiety for the target should not be greater than about 1×10⁻³M).

It will be appreciated by those of ordinary skill in the art thatnucleic acids in accordance with the present invention may comprisenucleotides entirely of the types found in naturally occurring nucleicacids, or may instead include one or more nucleotide analogs or have astructure that otherwise differs from that of a naturally occurringnucleic acid. U.S. Pat. Nos. 6,403,779; 6,399,754; 6,225,460; 6,127,533;6,031,086; 6,005,087; 5,977,089; and references therein disclose a widevariety of specific nucleotide analogs and modifications that may beused. See Crooke, S. (ed.) Antisense Drug Technology: Principles,Strategies, and Applications (1^(st) ed), Marcel Dekker; ISBN:0824705661; 1st edition (2001) and references therein. For example,2′-modifications include halo, alkoxy and allyloxy groups. In someembodiments, the 2′-OH group is replaced by a group selected from H, OR,R, halo, SH, NH₂, NR₂ or CN, wherein R is C₁-C₆ alkyl, alkenyl, oralkynyl, and halo is F, Cl, Br or I. Examples of modified linkagesinclude phosphorothioate and 5′-N-phosphoramidite linkages.

Nucleic acids of the present invention may include natural nucleosides(i.e., adenosine, thymidine, guanosine, cytidine, uridine,deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) ormodified nucleosides. Examples of modified nucleotides include basemodified nucleosides (e.g., aracytidine, inosine, isoguanosine,nebularine, pseudouridine, 2,6-diaminopurine, 2-aminopurine,2-thiothymidine, 3-deaza-5-azacytidine, 2′-deoxyuridine, 3-nitorpyrrole,4-methylindole, 4-thiouridine, 4-thiothymidine, 2-aminoadenosine,2-thiothymidine, 2-thiouridine, 5-bromocytidine, 5-iodouridine, inosine,6-azauridine, 6-chloropurine, 7-deazaadenosine, 7-deazaguanosine,8-azaadenosine, 8-azidoadenosine, benzimidazole, M1-methyladenosine,pyrrolo-pyrimidine, 2-amino-6-chloropurine, 3-methyl adenosine,5-propynylcytidine, 5-propynyluridine, 5-bromouridine, 5-fluorouridine,5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemically orbiologically modified bases (e.g., methylated bases), modified sugars(e.g., 2′-fluororibose, 2′-aminoribose, 2′-azidoribose,2′-O-methylribose, L-enantiomeric nucleosides arabinose, and hexose),modified phosphate groups (e.g., phosphorothioates andS′-N-phosphoramidite linkages), and combinations thereof. Natural andmodified nucleotide monomers for the chemical synthesis of nucleic acidsare readily available. In some cases, nucleic acids comprising suchmodifications display improved properties relative to nucleic acidsconsisting only of naturally occurring nucleotides. In some embodiments,nucleic acid modifications described herein are utilized to reduceand/or prevent digestion by nucleases (e.g. exonucleases, endonucleases,etc.). For example, the structure of a nucleic acid may be stabilized byincluding nucleotide analogs at the 3′ end of one or both strands orderto reduce digestion.

Modified nucleic acids need not be uniformly modified along the entirelength of the molecule. Different nucleotide modifications and/orbackbone structures may exist at various positions in the nucleic acid.One of ordinary skill in the art will appreciate that the nucleotideanalogs or other modification(s) may be located at any position(s) of anucleic acid such that the function of the nucleic acid is notsubstantially affected. To give but one example, modifications may belocated at any position of an aptamer such that the ability of theaptamer to specifically bind to the aptamer target is not substantiallyaffected. The modified region may be at the 5′-end and/or the 3′-end ofone or both strands. For example, modified aptamers in whichapproximately 1-5 residues at the 5′ and/or 3′ end of either ofemployed. The modification may be a 5′ or 3′ terminal modification. Oneor both nucleic acid strands may comprise at least 50% unmodifiednucleotides, at least 80% unmodified nucleotides, at least 90%unmodified nucleotides, or 100% unmodified nucleotides.

Nucleic acids in accordance with the present invention may, for example,comprise a modification to a sugar, nucleoside, or internucleosidelinkage such as those described in U.S. Patent Publications2003/0175950, 2004/0192626, 2004/0092470, 2005/0020525, and2005/0032733. The present invention encompasses the use of any nucleicacid having any one or more of the modification described therein. Forexample, a number of terminal conjugates, e.g., lipids such ascholesterol, lithocholic acid, aluric acid, or long alkyl branchedchains have been reported to improve cellular uptake. Analogs andmodifications may be tested using, e.g., using any appropriate assayknown in the art, for example, to select those that result in improveddelivery of a therapeutic agent, improved specific binding of an aptamerto an aptamer target, etc. In some embodiments, nucleic acids inaccordance with the present invention may comprise one or morenon-natural nucleoside linkages. In some embodiments, one or moreinternal nucleotides at the 3′-end, 5′-end, or both 3′- and 5′-ends ofthe aptamer are inverted to yield a such as a 3′-3′ linkage or a 5′-5′linkage.

Protein Targeting Moieties

In some embodiments, a targeting moiety in accordance with the presentinvention may be a protein or peptide. In certain embodiments, peptidesrange from about 5 to 100, 10 to 75, 15 to 50, or 20 to 25 amino acidsin size. In some embodiments, a peptide sequence a random arrangement ofamino acids.

The terms “polypeptide” and “peptide” are used interchangeably herein,with “peptide” typically referring to a polypeptide having a length ofless than about 100 amino acids. Polypeptides may contain L-amino acids,D-amino acids, or both and may contain any of a variety of amino acidmodifications or analogs known in the art. Useful modifications include,e.g., terminal acetylation, amidation, lipidation, phosphorylation,glycosylation, acylation, farnesylation, sulfation, etc.

In another embodiment, the targeting moiety can be a targeting peptideor targeting peptidomimetic has a length of at most 50 residues. In afurther embodiment, a nanoparticle of the invention contains a targetingpeptide or peptidomimetic that includes the amino acid sequence AKERC(SEQ ID NO: 3), CREKA (SEQ ID NO: 4), ARYLQKLN (SEQ ID NO: 5) orAXYLZZLN (SEQ ID NO: 6), wherein X and Z are variable amino acids, orconservative variants or peptidomimetics thereof. In particularembodiments, the targeting moiety is a peptide that includes the aminoacid sequence AKERC (SEQ ID NO: 3), CREKA. (SEQ ID NO: 4), ARYLQKLN (SEQID NO: 5) or AXYLZZLN (SEQ ID NO: 6), wherein X and Z are variable aminoacids, and has a length of less than 20, 50 or 100 residues. The CREKApeptide is known in the art, and is described in U.S. Patent ApplicationNo. 2005/0048063, which is incorporated herein by reference in itsentirety. The octapeptide AXYLZZLN (SEQ ID NO: 6) is described in Dinklaet al., The Journal of Biological Chemistry, Vol. 282, No. 26, pp.18686-18693, which is incorporated herein by reference in its entirety.

In one embodiment, the targeting moiety is an isolated peptide orpeptidomimetic that has a length of less than 100 residues and includesthe amino acid sequence CREKA (Cys Arg Glu Lys Ala.) or a peptidomimeticthereof. Such an isolated peptide- or peptidomimetic can have, forexample, a length of less than 50 residues or a length of less than 20residues. In particular embodiments, the invention provides a peptidethat includes the amino acid sequence CREKA and has a length of lessthan 20, 50 or 100 residues.

Moreover, the authors of The Journal of Biological Chemistry, Vol. 282,No. 26, pp. 18686-18693 describe a binding motif in streptococci thatforms an autoantigenic complex with human collagen IV. Accordingly, anypeptide, or conservative variants or peptidomimetics thereof, that bindsor forms a complex with collagen IV, or the basement membrane of a bloodvessel, can be used as a targeting moiety for the nanoparticles of theinvention.

Exemplary proteins that may be used as targeting moieties in accordancewith the present invention include, but are not limited to, antibodies,receptors, cytokines, peptide hormones, proteins derrived fromcombinatorial libraries (e.g. avimers, affibodies, etc.), andcharacteristic portions thereof.

In some embodiments, any protein targeting moiety can be utilized inaccordance with the present invention. To give but a few examples, IL-2,transferrin, GM-CSF, a-CD25, a-CD22, TGF-a, folic acid, a-CEA, a-EpCAMscFV, VEGF, LHRH, bombesin, somatostin, Gal, α-GD2, α-EpCAM, α-CD20,M0v19, scFv, α-Her-2, and α-CD64 can be used to target a variety ofcancers, such as lymphoma, glioma, leukemia, brain tumors, melanoma,ovarian cancer, neuroblastoma, folate receptor-expressing tumors,CEA-expressing tumors, EpCAM-expressing tumors, VEGF-expressing tumors,etc. (Eklund et al, 2005, Expert Rev. Anticancer Ther., 5:33; Kreitmanet al., 2000, J. Clin. OncoL, 18:1622; Kreitman et al, 2001, N. Engl. J.Med, 345:241; Sampson et al., 2003, J. Neurooncol, 65:27; Weaver et al.,2003, J. Neurooncol, 65:3; Leamon et al., 1993, J. Biol. Chem.,268:24847; Leamon et al., 1994, J. Drug Target., 2:101; Atkinson et al.,2001, J. Biol. Chem., 276:27930; Frankel et al., 2002, Clin. CancerRes., 8:1004; Francis et al, 2002, Br. J. Cancer, 87:600; de Graaf etal., 2002, Br. J. Cancer, 86:811; Spooner et al., 2003, Br. J. Cancer,88:1622; Liu et al, 1999, J. Drug Target., 7:43; Robinson et al, 2004,Proc. Natl. Acad. Sci., USA, 101:14527; Sondel et al, 2003, Curr. Opin.Investig. Drugs, 4:696; Connor et al., 2004, J. Immunother., 27:211;Gillies et al, 2005, Blood, 105:3972; Melani et al, 1998, Cancer Res.,58:4146; Metelitsa et al, 2002, Blood, 99:4166; Lyu et al, 2005, MolCancer Ther., 4:1205; and Hotter et al, 2001, Blood, 97:3138).

In some embodiments, protein targeting moieties can be peptides. One ofordinary skill in the art will appreciate that any peptide thatspecifically binds to a desired target can be used in accordance withthe present invention. In some embodiments, peptides targeting tumorvasculature are antagonists or inhibitors of angiogenic proteins thatinclude VEGFR (Binetruy-Tournaire et al, 2000, EMBO J., 19:1525), CD36(Reiher et al, 2002, Int. J. Cancer, 98:682) and Kumar et al, 2001,Cancer Res., 61:2232) aminopeptidase N (Pasqualini et al, 2000, CancerRes., 60:722), and matrix metalloproteinases (Koivunen et al., 1999,Nat. Biotechnol, 17:768). For instance, ATWLPPR (SEQ ID NO: 7) peptideis a potent antagonist of VEGF (Binetruy-Tournaire et al, 2000, EMBO j,19:1525); thrombospondin-1 (TSP-1) mimetics can induce apoptosis inendothelial cells (Reiher et al, 2002, Int. J. Cancer, 98:682);ROD-motif mimics (e.g. cyclic peptide ACDCRGDCFCG (SEQ ID NO: 8) and RODpeptidomimetic SCH 221153) block integrin receptors (Koivunen et al,1995, Biotechnology (NY), 13:265; and Kumar et al, 2001, Cancer Res.,61:2232); NGR-containing peptides (e.g. cyclic CNGRC (SEQ ID NO: 9))inhibit aminopeptidase N (Pasqualini et al, 2000, Cancer Res., 60:722);and cyclic peptides containing the sequence of HWGF (SEQ ID NO: 10)(e.g. CTTHWGFTLC (SEQ ID NO: 11)) selectively inhibit MMP-2 and MMP-9(Koivunen et al., 1999, Nat. Biotechnol., 17:768); and a LyP-1 peptidehas been identified (CGNKRTRGC (SEQ ID NO: 12)) which specifically bindsto tumor lymphatic vessels and induces apoptosis of endothelial cells(Laakkonen et al, 2004, Proc. Nail Acad. Sci., USA, 101:9381).

In some embodiments, peptide targeting moieties include peptide analogsthat block binding of peptide hormones to receptors expressed in humancancers (Bauer et al., 1982, Life Sci., 31:1133). Exemplary hormonereceptors (Reubi et al, 2003, Endocr. Rev., 24:389) include (1)somatostatin receptors (e.g. octreotide, vapreotide, and lanretode)(Froidevaux et al, 2002, Biopolymers, 66:161); (2)bombesin/gastrin-releasing peptide (GRP) receptor (e.g. RC-3940 series)(Kanashiro et al, 2003, Proc. Natl. Acad. Sci., USA, 100:15836); and (3)LHRH receptor (e.g. Decapeptyf, Lupron®, Zoladex®, and Cetrorelix®)(Schally et al., 2000, Prostate, 45:158).

In some embodiments, peptides that recognize IL-11 receptor-a can beused to target cells associated with prostate cancer tumors (see, e.g.,U.S. Patent Publication 2005/0191294).

In some embodiments, a targeting moiety may be an antibody and/orcharacteristic portion thereof. The term “antibody” refers to anyimmunoglobulin, whether natural or wholly or partially syntheticallyproduced and to derivatives thereof and characteristic portions thereof.An antibody may be monoclonal or polyclonal. An antibody may be a memberof any immunoglobulin class, including any of the human classes: IgG,IgM, IgA, IgD, and IgE. One of ordinary skill in the art will appreciatethat any antibody that specifically binds to a desired target can beused in accordance with the present invention.

In some embodiments, antibodies that recognize PSMA can be used totarget cells associated with prostate cancer tumors. Such antibodiesinclude, but are not limited to, scFv antibodies A5, GO, G1, G2, and G4and mAbs 3/B7, 3/F11, 3/A12, K7, K12, and D20 (Elsasser-Beile et al,2006, Prostate, 66:1359); mAbs E99, J591, J533, and J415 (Liu et al,1997, Cancer Res., 57:3629; Liu et al, 1998, Cancer Res., 58:4055;Fracasso et al., 2002, Prostate, 53:9; McDevitt et al, 2000, CancerRes., 60:6095; McDevitt et al., 2001, Science, 294:1537; Smith-Jones etal, 2000, Cancer Res., 60:5237; Vallabhajosula of al., 2004, Prostate,58:145; Bander er a/., 2003, J. C/ro/., 170:1717; Patri et al., 2004,Bioconj. Chem., 15:1174; and U.S. Pat. No. 7,163,680); mAb 7E11-05.3(Horoszewicz et al., 1987, Anticancer Res., 7:927); antibody 7E11(Horoszewicz et al, 1987, Anticancer Res., 7:927; and U.S. Pat. No.5,162,504); and antibodies described in Chang et al, 1999, Cancer Res.,59:3192; Murphy et al., 1998, J. UroL, 160:2396; Grauer et al, 1998,Cancer Res., 58:4787; and Wang era/., 2001, M J. Cancer, 92:871. One ofordinary skill in the art will appreciate that any antibody thatrecognizes and/or specifically binds to PSMA may be used in accordancewith the present invention.

In some embodiments, antibodies which recognize other prostatetumor-associated antigens are known in the art and can be used inaccordance with the present invention to target cells associated withprostate cancer tumors (see, e.g., Vihko et al, 1985, Biotechnology inDiagnostics, 131; Babaian et al, 1987, J. UroL, 137:439; Leroy et al.,1989, Cancer, 64:1; Meyers et al, 1989, Prostate, 14:209; and U.S. Pat.Nos. 4,970,299; 4,902,615; 4,446,122 and Re 33,405; 4,862,851;5,055,404). To give but a few examples, antibodies have been identifiedwhich recognize transmembrane protein 24P4C12 (U.S. Patent Publication2005/0019870); calveolin (U.S. Patent Publications 2003/0003103 and2001/0012890); L6 (U.S. Patent Publication 2004/0156846); prostatespecific reductase polypeptide (U.S. Pat. No. 5,786,204; and U.S. PatentPublication 2002/0150578); and prostate stem cell antigen (U.S. PatentPublication 2006/0269557).

In some embodiments, protein targeting moieties that may be used totarget cells associated with prostate cancer tumors includeconformationally constricted dipeptide mimetics (Ding et al, 2004, Org.Lett., 6:1805).

As used herein, an antibody fragment (i.e., characteristic portion of anantibody) refers to any derivative of an antibody which is less thanfull-length. In general, an antibody fragment retains at least asignificant portion of the full-length antibody's specific bindingability. Examples of antibody fragments include, but are not limited to,Fab, Fab″, F(ab′)2, scFv, Fv, dsFv diabody, and Fd fragments.

An antibody fragment can be produced by any means. For example, anantibody fragment may be enzymatically or chemically produced byfragmentation of an intact antibody and/or it may be recombinantlyproduced from a gene encoding the partial antibody sequence.Alternatively or additionally, an antibody fragment may be wholly orpartially synthetically produced. An antibody fragment may optionallycomprise a single chain antibody fragment. Alternatively oradditionally, an antibody fragment may comprise multiple chains that arelinked together, for example, by disulfide linkages. An antibodyfragment may optionally comprise a multimolecular complex. A functionalantibody fragment will typically comprise at least about 50 amino acidsand more typically will comprise at least about 200 amino acids. In someembodiments, antibodies may include chimeric (e.g., “humanized”) andsingle chain (recombinant) antibodies. In some embodiments, antibodiesmay have reduced effector functions and/or bispecific molecules. In someembodiments, antibodies may include fragments produced by a Fabexpression library.

Single-chain Fvs (scFvs) are recombinant antibody fragments consistingof only the variable light chain (VL) and variable heavy chain (VH)covalently connected to one another by a polypeptide linker. Either VLor VH may comprise the NH₂-terminal domain. The polypeptide linker maybe of variable length and composition so long as the two variabledomains are bridged without significant steric interference. Typically,linkers primarily comprise stretches of glycine and serine residues withsome glutamic acid or lysine residues interspersed for solubility.

Diabodies are dimeric scFvs. Diabodies typically have shorter peptidelinkers than most scFvs, and they often show a preference forassociating as dimers.

An Fv fragment is an antibody fragment which consists of one VH and oneVL domain held together by noncovalent interactions. The term “dsFv” asused herein refers to an Fv with an engineered intermolecular disulfidebond to stabilize the VH-VL pair.

A Fab′ fragment is an antibody fragment essentially equivalent to thatobtained by reduction of the disulfide bridge or bridges joining the twoheavy chain pieces in the F(ab′)2 fragment. The Fab′ fragment may berecombinantly produced.

A Fab fragment is an antibody fragment essentially equivalent to thatobtained by digestion of immunoglobulins with an enzyme (e.g. papain).The Fab fragment may be recombinantly produced. The heavy chain segmentof the Fab fragment is the Fd piece.

Carbohydrate Targeting Moieties

In some embodiments, a targeting moiety in accordance with the presentinvention may comprise a carbohydrate. To give but one example, lactoseand/or galactose can be used for targeting hepatocytes.

In some embodiments, a carbohydrate may be a polysaccharide comprisingsimple sugars (or their derivatives) connected by glycosidic bonds, asknown in the art. Such sugars may include, but are not limited to,glucose, fructose, galactose, ribose, lactose, sucrose, maltose,trehalose, cellbiose, mannose, xylose, arabinose, glucdronic acid,galactoronic acid, mannuronic acid, glucosamine, galatosatnine, andneuramic acid. In some embodiments, a carbohydrate may be one or more ofpullulan, cellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, hydroxycellulose, methylcellulose, dextran,cyclodextran, glycogen, starch, hydroxyethylstarch, carageenan, glycon,amylose, chitosan, algin and alginic acid, starch, chitin, heparin,konjac, glucommannan, pustulan, heparin, hyaluronic acid, curdlan, andxanthan.

In some embodiments, the carbohydrate may be aminated, carboxylated,and/or sulfated. In some embodiments, hydropbilic polysaccharides can bemodified to become hydrophobic by introducing a large number ofside-chain hydrophobic groups. In some embodiments, a hydrophobiccarbohydrate may include cellulose acetate, pullulan acetate, konjacacetate, amylose acetate, and dextran acetate.

Lipid Targeting Moieties

In some embodiments, a targeting moiety in accordance with the presentinvention may comprise one or more fatty acid groups or salts thereof.In some embodiments, a fatty acid group may comprise digestible, longchain (e.g., C₈-C₅₀), substituted or unsubstituted hydrocarbons. In someembodiments, a fatty acid group may be a C₁₀-C₂₀ fatty acid or saltthereof. In some embodiments, a fatty acid group may be a C₁₅-C₂₀ fattyacid or salt thereof. In some embodiments, a fatty acid group may beunsaturated. In some embodiments, a fatty acid group may bemonounsaturated. In some embodiments, a fatty acid group may bepolyunsaturated. In some embodiments, a double bond of an unsaturatedfatty acid group may be in the cis conformation. In some embodiments, adouble bond of an unsaturated fatty acid may be in the transconformation.

In some embodiments, a fatty acid group may be one or more of butyric,caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

The targeting moiety can be conjugated to the polymeric matrix oramphiphilic component using any suitable conjugation technique. Forinstance, two polymers such as a targeting moiety and a biocompatiblepolymer, a biocompatible polymer and a poly(ethylene glycol), etc., maybe conjugated together using techniques such as EDC-NHS chemistry(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride andN-hydroxysuccinimide) or a reaction involving a maleimide or acarboxylic acid, which can be conjugated to one end of a thiol, anamine, or a similarly functionalized polyether. The conjugation of suchpolymers, for instance, the conjugation of a poly(ester) and apoly(ether) to form a poly(ester-ether), can be performed in an organicsolvent, such as, but not limited to, dichloromethane, acetonitrile,chloroform, dimethylformamide, tetrahydrofuran, acetone, or the like.Specific reaction conditions can be determined by those of ordinaryskill in the art using no more than routine experimentation.

In another set of embodiments, a conjugation reaction may be performedby reacting a polymer that comprises a carboxylic acid functional group(e.g., a poly(ester-ether) compound) with a polymer or other moiety(such as a targeting moiety) comprising an amine. For instance, atargeting moiety, such as an aptamer or peptide, may be reacted with anamine to form an amine-containing moiety, which can then be conjugatedto the carboxylic acid of the polymer. Such a reaction may occur as asingle-step reaction, i.e., the conjugation is performed without usingintermediates such as N-hydroxysuccinimide or a maleimide. Theconjugation reaction between the amine-containing moiety and thecarboxylic acid-terminated polymer (such as a poly(ester-ether)compound) may be achieved, in one set of embodiments, by adding theamine-containing moiety, solubilized in an organic solvent such as (butnot limited to) dichloromethane, acetonitrile, chloroform,tetrahydrofuran, acetone, formamide, dimethylformamide, pyridines,dioxane, or dimethysulfoxide, to a solution containing the carboxylicacid-terminated polymer. The carboxylic acid-terminated polymer may becontained within an organic solvent such as, but not limited to,dichloromethane, acetonitrile, chloroform, dimethylformamide,tetrahydrofuran, or acetone. Reaction between the amine-containingmoiety and the carboxylic acid-terminated polymer may occurspontaneously, in some cases. Unconjugated reactants may be washed awayafter such reactions, and the polymer may be precipitated in solventssuch as, for instance, ethyl ether, hexane, methanol, or ethanol.

A polymeric conjugate (i.e., a targeting moiety covalently bound to thepolymeric component of the reverse micelle) of the present invention maybe formed using any suitable conjugation technique. For instance, twocompounds such as a targeting moiety and a biocompatible polymer, abiocompatible polymer and a poly(ethylene glycol), etc., may beconjugated together using techniques such as EDC-NHS chemistry(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride andN-hydroxysuccinimide) or a reaction involving a maleimide or acarboxylic acid, which can be conjugated to one end of a thiol, anamine, or a similarly functionalized polyether. The conjugation of suchpolymers, for instance, the conjugation of a poly(ester) and apoly(ether) to form a poly(ester-ether), can be performed in an organicsolvent, such as, but not limited to, dichloromethane, acetonitrile,chloroform, dimethylformamide, tetrahydrofuran, acetone, or the like.Specific reaction conditions can be determined by those of ordinaryskill in the art using no more than routine experimentation.

In another set of embodiments, a conjugation reaction may be performedby reacting a polymer that comprises a carboxylic acid functional group(e.g., a poly(ester-ether) compound) with a polymer or other moiety(such as a targeting moiety) comprising an amine. For instance, atargeting moiety, such as an aptamer, may be reacted with an amine toform an amine-containing moiety, which can then be conjugated to thecarboxylic acid of the polymer. Such a reaction may occur as asingle-step reaction, i.e., the conjugation is performed without usingintermediates such as N-hydroxysuccinimide or a maleimide. Theconjugation reaction between the amine-containing moiety (e.g., aptamer)and the carboxylic acid-terminated polymer (such as a poly(ester-ether)compound) may be achieved, in one set of embodiments, by adding theamine-containing moiety, solubilized in an organic solvent such as (butnot limited to) dichloromethane, acetonitrile, chloroform,tetrahydrofuran, acetone, formamide, dimethylformamide, pyridines,dioxane, or dimethysulfoxide, to a solution containing the carboxylicacid-terminated polymer. The carboxylic acid-terminated polymer may becontained within an organic solvent such as, but not limited to,dichloromethane, acetonitrile, chloroform, dimethylformamide,tetrahydrofuran, or acetone. Reaction between the amine-containingmoiety and the carboxylic acid-terminated polymer may occurspontaneously, in some cases. Unconjugated reactants may be washed awayafter such reactions, and the polymer may be precipitated in solventssuch as, for instance, ethyl ether, hexane, methanol, or ethanol.

As a specific example, an aptamer may be prepared as a targeting moietyin a particle as follows. Carboxylic acid modifiedpoly(lactide-co-glycolide) (PLGA-COOH) may be conjugated to anamine-modified heterobifunctional poly(ethylene glycol) (NH₂-PEG-COOH)to form a copolymer of PLGA-PEG-COOH. By using either an aminecontaining or an amine-modified aptamer (NH₂-Apt), a triblock polymer ofPLGA-PEG-Apt may be formed by conjugating the carboxylic acid end of thePEG to the amine functional group on the aptamer. The multiblock polymercan then be used, for instance, as discussed below, e.g., fortherapeutic applications.

Preparation of Polymer-Encapsulated Reverse Micelles

Another aspect of the invention is directed to systems and methods ofproducing such polymer-encapsulated reverse micelles. As mentioned, oneaspect of the invention is directed to a method of developingpolymer-encapsulated reverse micelles with desired properties, such asdesired chemical, biological, or physical properties. In one set ofembodiments, the method includes producing libraries ofpolymer-encapsulated reverse micelles having highly controlledproperties, which can be formed by mixing together two or more polymersin different ratios. By mixing together two or more different polymers(e.g., copolymers, e.g., block copolymers) in different ratios andproducing particles from the polymers (e.g., copolymers, e.g., blockcopolymers), particles having highly controlled properties may beformed. For example, one polymer (e.g., copolymer, e.g., blockcopolymer) may include a targeting moiety, while another polymer (e.g.,copolymer, e.g., block copolymer) may be chosen for its biocompatibilityand/or its ability to control immunogenicity of the resultantpolymer-encapsulated reverse micelles.

Two major methods of forming nanoparticles that encapsulate nucleicacids currently exist. These involve: (1) forming water/oil/water doubleemulsions, which have the effect of dispersing the hydrophilic agentthroughout the phase containing the polymer prior to polymerprecipitation and nanoparticle formation using high frequency sonicationand/or honiogenization, and (2) using unnatural cationic emulsifiers andcondensing agents, typically cationic lipids, cationic polymers, orcationic small molecules, to disperse the negatively charged nucleicacid in the phase containing the polymer.

These methods are disfavored for a variety of reasons. In the case ofthe first method, the major limitations are: (1) loss of nucleic acidactivity due to high shear denaturation and molecule damage, (2) lowreproducibility of results, (3) low encapsulation yield, particularlyfor low molecular weight nucleic acids, and (4) low nucleic acid contentper nanoparticle. These issues are a consequence of the physicochemicalcharacteristics of the polymer and the nucleic acid, and as such, aredifficult to overcome. In the case of the second method, the toxicity ofcationic polymers, lipids, and small molecules have been welldocumented. These systems are disfavored due to the toxicity of thecomponents, which are capable of non-selectively damaging the biologicalmilieu of interest. In addition, this approach does not appreciate theeffect of forming reverse micelles prior to the addition of polymer,which can potentially cause an undesirable competition in thenanoparticle formation process.

The disclosed invention overcomes the limitations imposed by the firstmethod by effectively dispersing the therapeutic agent (e.g., nucleicacid) of interest into a fine nanosuspension of reverse micelles priorto nanoparticle formation, without the use of high frequency sonicationor homogenization. The reverse micellization step effectively packagestherapeutic agents (e.g., nucleic acids) by improving the thermodynamicsof interaction between the nucleic acids and the biodegradable polymer,thereby improving the encapsulation yield and nucleic acid content pernanoparticle. The disclosed invention requires no high frequencysonication or homogenization, and therefore does not incur the lossesthat are common in methods that incorporate those techniques. Thedisclosed invention overcomes the toxicity issues in using cationiclipids, polymers, and small molecules by using non-toxic and/ornaturally-occurring-uncharged and negatively charged lipids. The presentinvention involves forming reverse micelles prior to nanoprecipitation,thereby avoiding the potential competition that is a result of one-stepprocedures. The use of the nanoprecipitation method improves thereproducibility of results by making the desired nanoparticle athermodynamically favored result.

Thus, in one set of embodiments, the polymer-encapsulated reversemicelles are formed by providing one or more solutions comprising one ormore therapeutic agents (e.g., a nucleic acid) and amphiphiliccomponents, and contacting them with certain solvents to produce areverse micelle. A polymer is then added to the micelle solution, andthis solution is then added to a different solvent to form apolymer-encapsulated reverse micelle.

For example, the therapeutic agent (e.g., nucleic acid) to beencapsulated is first incorporated into reverse micelles by mixing theagent with naturally derived and non-toxic amphipathic lipids in avolatile, water-miscible organic solvent. In one embodiment, thetherapeutic agent to be encapsulated is a neucleic acid, including, butnot limited to, natural or unnatural siRNAs, shRNAs, microRNAs,ribozymes, DNA plasmids, aptamers, antisense oligonucleotides,randomized oligonucleotides, or ribozymes. The amphipathic lipid can be,but is not limited to, one or a plurality of the following:phosphatidylcholine, lipid A, cholesterol, dolichol, shingosine,sphingomyelin, ceramide, cerebroside, sulfatide, glycosylceramide,phytosphingosine, phosphatidylethanolamine, phosphatidylglycerol,phosphatidylinositol, phosphatidylserine, cardiolipin, phophatidic acid,and lysophophatides. The volatile, water-miscible organic solvent canbe, but is not limited to, tetrahydrofuran, acetone, acetonitrile, ordmethylformamide. The biodegradable polymer is added to this mixtureafter reverse micelle formation is complete. In a preferred embodiment,the biodegradable polymer can be, but is not limited to one or aplurality of the following: poly(D,L-lactic acid), poly(D,L-glycolicacid), poly(ε-caprolactone), their copolymers at various molar ratios,and their copolymers diblocked or multiblocked with poly(ethyleneglycol). The resulting biodegradable polymer-reverse micelle mixture iscombined with a polymer-insoluble hydrophilic non-solvent to formnanoparticles by the rapid diffusion of the solvent into the non-solventand evaporation of the organic solvent. In a preferred embodiment, thepolymer-insoluble hydrophilic non-solvent can be, but is not limited toone or a plurality of the following: water, ethanol, methanol, andmixtures thereof.

The methods of the invention can be used to create a library ofpolymer-encapsulated reverse micelles. By creating a library of suchparticles, particles having any desirable properties may be identified.For example, properties such as surface functionality, surface charge,size, zeta (ζ) potential, hydrophobicity, ability to controlimmunogenicity, and the like, may be highly controlled. For instance, alibrary of particles may be synthesized, and screened to identify theparticles having a particular ratio of polymers that allows theparticles to have a specific density of moieties (e.g., aptamers)present on the surface of the particle. This allows particles having oneor more specific properties to be prepared, for example, a specific sizeand a specific surface density of moieties, without an undue degree ofeffort. Accordingly, certain embodiments of the invention are directedto screening techniques using such libraries, as well as any particlesidentified using such libraries. In addition, identification may occurby any suitable method. For instance, the identification may be director indirect, or proceed quantitatively or qualitatively.

In another embodiment, the invention provides a method of preparing apolymer-encapsulated reverse micelle wherein the micelle has a ratio ofligand-bound polymer to non-functionalized polymer effective for thetreatment of a disease, e.g., cancer, e.g., prostate cancer, wherein thehydrophilic, ligand-bound polymer is conjugated to a lipid that willself assemble with the hydrophobic polymer, such that the hydrophobicand hydrophilic polymers that constitute the nanoparticle are notcovalently bound. “Self-assembly” refers to a process of spontaneousassembly of a higher order structure that relies on the naturalattraction of the components of the higher order structure (e.g.,molecules) for each other. It typically occurs through random movementsof the molecules and formation of bonds based on size, shape,composition, or chemical properties. For example, such a methodcomprises providing a first polymer that is reacted with a lipid, toform a polymer/lipid conjugate. The polymer/lipid conjugate is thenreacted with a targeting moiety (e.g., an aptamer) to prepare aligand-bound polymer/lipid conjugate; and mixing the ligand-boundpolymer/lipid conjugate with a second, non-functionalized polymer, andthe therapeutic agent; such that the stealth nanoparticle is formed. Incertain embodiments, the first polymer is PEG, such that alipid-terminated PEG is formed. In one embodiment, the lipid is of theFormula V, e.g., 2 distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),and salts thereof, e.g., the sodium salt. The lipid-terminated PEG canthen, for example, be mixed with PLGA to form a particle.

In some cases, a population of polymer-encapsulated reverse micelles maybe present. For example, a population of particles may include at least20 particles, at least 50 particles, at least 100 particles, at least300 particles, at least 1,000 particles, at least 3,000 particles, or atleast 10,000 particles. Various embodiments of the present invention aredirected to such populations of particles. For instance, in someembodiments, the particles may each be substantially the same shapeand/or size (“monodisperse”). For example, the particles may have adistribution of characteristic dimensions such that no more than about5% or about 10% of the particles have a characteristic dimension greaterthan about 10% greater than the average characteristic dimension of theparticles, and in some cases, such that no more than about 8%, about 5%,about 3%, about 1%, about 0.3%, about 0.1%, about 0.03%, or about 0.01%have a characteristic dimension greater than about 10% greater man theaverage characteristic dimension of the particles. In some cases, nomore than about 5% of the particles have a characteristic dimensiongreater than about 5%, about 3%, about 1%, about 0.3%, about 0.1%, about0.03%, or about 0.01% greater than the average characteristic dimensionof the particles.

The library of particles can then be screened in some fashion toidentify those polymer-encapsulated reverse micelles having one or moredesired properties, for example, surface functionality, surface charge,size, zeta (ζ) potential, hydrophobicity, ability to controlimmunogenicity, and the like. One or more of the macromolecules withinthe particles may include one or more polymers chosen to bebiocompatible or biodegradable, one or more polymers chosen to reduceimmunogenicity, and/or one or more targeting moieties. Themacromolecules within the library may comprise some or all of thesepolymers, in any suitable combination (including, but not limited to,combinations in which a first polymer comprises a targeting moiety and asecond polymer does not contain any of these species).

The nanoparticles described above may also contain therapeutic agents.Examples of therapeutic agents include, but are not limited to, achemotherapeutic agent, a radioactive agent, a nucleic acid-based agent,a lipid-based agent, a carbohydrate based agent, a natural smallmolecule, or a synthetic small molecule.

Therapeutic Agents

According to the present invention, any agents (“payload”), including,for example, therapeutic agents (e.g. anti-cancer agents), diagnosticagents (e.g. contrast agents; radionuclides; and fluorescent,luminescent, and magnetic moieties), prophylactic agents (e.g.vaccines), and/or nutraceutical agents (e.g. vitamins, minerals, etc.)may be delivered by the nanoparticles of the invention. Exemplary agentsto be delivered in accordance with the present invention include, butare not limited to, small molecules (e.g. cytotoxic agents), nucleicacids (e.g., siRNA, RNAi, and mircoRNA agents), proteins (e.g.antibodies), peptides, lipids, carbohydrates, hormones, metals,radioactive elements and compounds, drugs, vaccines, immunologicalagents, etc., and/or combinations thereof. In some embodiments, theagent to be delivered is an agent useful in the treatment of cancer(e.g., prostate cancer).

For instance, the targeting moiety may target or cause the particle tobecome localized at specific portions within a subject, and the payloadmay be delivered to those portions. In a particular embodiment, the drugor other payload is released in a controlled release manner from theparticle and allowed to interact locally with the particular targetingsite (e.g., a tumor). The term “controlled release” (and variants ofthat term) as used herein (e.g., in the context of “controlled-releasesystem”) is generally meant to encompass release of a substance (e.g., adrug) at a selected site or otherwise controllable in rate, interval,and/or amount. Controlled release encompasses, but is not necessarilylimited to, substantially continuous delivery, patterned delivery (e.g.,intermittent delivery over a period of time that is interrupted byregular or irregular time intervals), and delivery of a bolus of aselected substance (e.g., as a predetermined, discrete amount if asubstance over a relatively short period of time (e.g., a few seconds orminutes)).

For example, a targeting portion may cause the particles to becomelocalized to a tumor, a disease site, a tissue, an organ, a type ofcell, etc. within the body of a subject, depending on the targetingmoiety used. For example, a targeting moiety, e.g., an aptamer, maybecome localized to prostate cancer cells.

In particular embodiments, the agent to be delivered is a nucleic acidselected from the group consisting of natural or unnatural siRNAs,shRNAs, microRNAs, ribozymes, DNA plasmids, aptamers, antisenseoligonucleotides, randomized oligonucleotides, or ribozymes.

In one embodiment, the reverse micelle particles of this invention willcontain nucleic acids such as siRNA.

Preferably, the siRNA molecule has a length from about 10-50 or morenucleotides. More preferably, the siRNA molecule has a length from about15-45 nucleotides. Even more preferably, the siRNA molecule has a lengthfrom about 19-40 nucleotides. Even more preferably, the siRNA moleculehas a length of from about 21-23 nucleotides.

The siRNA of the invention preferably mediates RNAi against a targetmRNA. The siRNA molecule can be designed such that every residue iscomplementary to a residue in the target molecule. Alternatively, one ormore substitutions can be made within the molecule to increase stabilityand/or enhance processing activity of said molecule. Substitutions canbe made within the strand or can be made to residues at the ends of thestrand.

The target mRNA cleavage reaction guided by siRNAs is sequence specific.In general, siRNA containing a nucleotide sequence identical to aportion of the target gene are preferred for inhibition. However, 100%sequence identity between the siRNA and the target gene is not requiredto practice the present invention. Sequence variations can be toleratedincluding those that might be expected due to genetic mutation, strainpolymorphism, or evolutionary divergence. For example, siRNA sequenceswith insertions, deletions, and single point mutations relative to thetarget sequence have also been found to be effective for inhibition.Alternatively, siRNA sequences with nucleotide analog substitutions orinsertions can be effective for inhibition.

Moreover, not all positions of an siRNA contribute equally to targetrecognition. Mismatches in the center of the siRNA are most critical andessentially abolish target RNA cleavage. In contrast, the 3′ nucleotidesof the siRNA do not contribute significantly to specificity of thetarget recognition. Generally, residues at the 3′ end of the siRNAsequence which is complementary to the target RNA (e.g., the guidesequence) are not critical for target RNA cleavage.

Sequence identity may readily be determined by sequence comparison andalignment algorithms known in the art. To determine the percent identityof two nucleic acid sequences (or of two amino acid sequences), thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in the first sequence or second sequence for optimalalignment). The nucleotides (or amino acid residues) at correspondingnucleotide (or amino acid) positions are then compared. When a positionin the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=# of identical positions/total # ofpositions×100), optionally penalizing the score for the number of gapsintroduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Apreferred, non-limiting example of a local alignment algorithm utilizedfor the comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873. Such an algorithm isincorporated into the BLAST programs (version 2.0) of Altschul, et al.(1990) J Mol Biol. 215:403-10.

In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the length ofthe aligned sequences (i.e., a gapped alignment). To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389. Apreferred, non-limiting example of a mathematical algorithm utilized forthe global comparison of sequences is the algorithm of Myers and Miller,CABIOS (1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

Greater than 90% sequence identity, e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or even 100% sequence identity, between the siRNA and theportion of the target mRNA is preferred. Alternatively, the siRNA may bedefined functionally as a nucleotide sequence (or oligonucleotidesequence) that is capable of hybridizing with a portion of the targetmRNA transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C. or 70° C. hybridization for 12-16 hours; followed by washing).Additional hybridization conditions include hybridization at 70° C. in1×SSC or 50° C. in 1×SSC, 50% formamide followed by washing at 70° C. in0.3×SSC or hybridization at 70° C. in 4×SSC or 50° C. in 4×SSC, 50%formamide followed by washing at 67° C. in 1×SSC. The hybridizationtemperature for hybrids anticipated to be less than 50 base pairs inlength should be 5-10° C. less than the melting temperature (Tm) of thehybrid, where Tm is determined according to the following equations. Forhybrids less than 18 base pairs in length, Tm(° C.)=2(# of A+Tbases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs inlength, Tm(° C.)=81.5+16.6(log₁₀[Na+])+0.41 (% G+C)−(600/N), where N isthe number of bases in the hybrid, and [Na+] is the concentration ofsodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165 M).Additional examples of stringency conditions for polynucleotidehybridization are provided in Sambrook, J., E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11,and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al.,eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporatedherein by reference. The length of the identical nucleotide sequencesmay be at least about or about equal to 10, 12, 15, 17, 20, 22, 25, 27,30, 32, 35, 37, 40, 42, 45, 47 or 50 bases.

In one embodiment, the siRNA molecules of the present invention aremodified to improve stability in serum or in growth medium for cellcultures. In order to enhance the stability, the 3′-residues may bestabilized against degradation, e.g., they may be selected such thatthey consist of purine nucleotides, particularly adenosine or guanosinenucleotides. Alternatively, substitution of pyrimidine nucleotides bymodified analogues, e.g., substitution of uridine by 2′-deoxythymidineis tolerated and does not affect the efficiency of RNA interference. Forexample, the absence of a 2′ hydroxyl may significantly enhance thenuclease resistance of the siRNAs in tissue culture medium.

In another embodiment of the present invention the siRNA molecule maycontain at least one modified nucleotide analogue. The nucleotideanalogues may be located at positions where the target-specificactivity, e.g., the RNAi mediating activity is not substantiallyeffected, e.g., in a region at the 5′-end and/or the 3′-end of the RNAmolecule. Particularly, the ends may be stabilized by incorporatingmodified nucleotide analogues.

Nucleotide analogues include sugar- and/or backbone-modifiedribonucleotides (i.e., include modifications to the phosphate-sugarbackbone). For example, the phosphodiester linkages of natural RNA maybe modified to include at least one of a nitrogen or sulfur heteroatom.In preferred backbone-modified ribonucleotides the phosphoester groupconnecting to adjacent ribonucleotides is replaced by a modified group,e.g., of phosphothioate group. In preferred sugar modifiedribonucleotides, the 2′OH-group is replaced by a group selected from H,OR, R, halo, SH, SR, NH₂, NHR, NR₂ or NO₂, wherein R is C1-C6 alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I.

Nucleotide analogues also include nucleobase-modified ribonucleotides,i.e., ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. Itshould be noted that the above modifications may be combined.

RNA may be produced enzymatically or by partial/total organic synthesis,any modified ribonucleotide can be introduced by in vitro enzymatic ororganic synthesis. In one embodiment, an siRNA is prepared chemically.Methods of synthesizing RNA molecules are known in the art, inparticular, the chemical synthesis methods as described in Verina andEckstein (1998), Annul Rev. Biochem. 67:99. In another embodiment, ansiRNA is prepared enzymatically. For example, an siRNA can be preparedby enzymatic processing of a long, double-stranded RNA having sufficientcomplementarity to the desired target mRNA. Processing of long RNA canbe accomplished in vitro, for example, using appropriate cellularlysates and siRNAs can be subsequently purified by gel electrophoresisor gel filtration. siRNA can then be denatured according toart-recognized methodologies. In an exemplary embodiment, siRNA can bepurified from a mixture by extraction with a solvent or resin,precipitation, electrophoresis, chromatography, or a combinationthereof. Alternatively, the siRNA may be used with no or a minimum ofpurification to avoid losses due to sample processing.

Alternatively, the siRNAs can also be prepared by enzymatictranscription from synthetic DNA templates or from DNA plasmids isolatedfrom recombinant bacteria. Typically, phage RNA polymerases are usedsuch as T7, T3 or SP6 RNA polyimerase (Milligan and Uhlenbeck (1989)Methods Enzyniol 180:51-62). The RNA may be dried for storage ordissolved in an aqueous solution. The solution may contain buffers orsalts to inhibit annealing, and/or promote stabilization of the doublestrands.

Commercially available design tools and kits, such as those availablefrom Ambion, Inc. (Austin, Tex.), and the Whitehead Institute ofBiomedical Research at MIT (Cambridge, Mass.) allow for the design andproduction of siRNA. By way of example, a desired mRNA sequence can beentered into a sequence program that will generate sense and antisensetarget strand sequences. These sequences can then be entered into aprogram that determines the sense and antisense siRNA oligonucleotidetemplates. The programs can also be used to add, e.g., hairpin insertsor T1 promoter primer sequences. Kits also can then be employed to buildsiRNA expression cassettes.

In various embodiments, siRNAs are synthesized in vivo, in situ, and invitro. Endogenous RNA polymerase of the cell may mediate transcriptionin vivo or in situ, or cloned RNA polymerase can be used fortranscription in vivo or in vitro. For transcription from a transgene invivo or an expression construct, a regulatory region (e.g., promoter,enhancer, silencer, splice donor and acceptor, polyadenylation) may beused to transcribe the siRNAs. Inhibition may be targeted by specifictranscription in an organ, tissue, or cell type; stimulation of anenvironmental condition (e.g., infection, stress, temperature, chemicalinducers); and/or engineering transcription at a developmental stage orage. A transgenic organism that expresses siRNAs from a recombinantconstruct may be produced by introducing the construct into a zygote, anembryonic stem cell, or another multipotent cell derived from theappropriate organism.

In one embodiment, the target mRNA of the invention specifies the aminoacid sequence of at least one protein such as a cellular protein (e.g.,a nuclear, cytoplasmic, transmembrane, or membrane-associated protein).In another embodiment, the target mRNA of the invention specifies theamino acid sequence of an extracellular protein (e.g., an extracellularmatrix protein or secreted protein). As used herein, the phrase“specifies the amino acid sequence” of a protein means that the mRNAsequence is translated into the amino acid sequence according to therules of the genetic code. The following classes of proteins are listedfor illustrative purposes: developmental proteins (e.g., adhesionmolecules, cyclin kinase inhibitors, Wnt family members, Pax familymembers, Winged helix family members, Hox family members,cytokines/lymphokines and their receptors, growth/differentiationfactors and their receptors, neurotransmitters and their receptors);oncogene-encoded proteins (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2. CBL,CSFIR, ERBA, ERBB, EBRB2, ERBB2, ERBB3, ETSI, ETSI, ETV6, FGR, FOS, FYN,HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS,PIM 1, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressor proteins(e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF 1, NF2, RB 1, TP53, and WTI);and enzymes (e.g., ACC synthases and oxidases, ACP desaturases andhydroxylases, ADPglucose pyrophorylases, acetylases and deacetylases,ATPases, alcohol dehydrogenases, amylases, amyloglucosidases, catalases,cellulases, chalcone synthases, chitinases, cyclooxygenases,decarboxylases, dextrinases, DNA and RNA polymerases, galactosidases,glucanases, glucose oxidases, granule-bound starch synthases, GTPases,helicases, hemicellulases, integrases, inulinases, invertases,isomerases, kinases, lactases, lipases, lipoxygenases, lysozymes,nopaline synthases, octopine synthases, pectinesterases, peroxidases,phosphatases, phospholipases, phosphorylases, phytases, plant growthregulator synthases, polygalacturonases, proteinases and peptidases,pullanases, recombinases, reverse transcriptases, RUBISCOs,topoisomerases, and xylanases), proteins involved in tumor growth(including vascularization) or in metastatic activity or potential,including cell surface receptors and ligands as well as secretedproteins, cell cycle regulatory, gene regulatory, and apoptosisregulatory proteins, immune response, inflammation, complement, orclotting regulatory proteins.

As used herein, the term “oncogene” refers to a gene which stimulatescell growth and, when its level of expression in the cell is reduced,the rate of cell growth is reduced or the cell becomes quiescent. In thecontext of the present invention, oncogenes include intracellularproteins, as well as extracellular growth factors which may stimulatecell proliferation through autocrine or paracrine function. Examples ofhuman oncogenes against which siRNA and morpholino constructs candesigned include c-myc, c-myb, mdm2, PKA-I (protein kinase A type I),Abl-1, Bcl2, Ras, c-Raf kinase, CDC25 phosphatases, cyclins, cyclindependent kinases (cdks), telomerase, PDGF/sis, erb-B, fos, jun, mos,and src, to name but a few. In the context of the present invention,oncogenes also include a fusion gene resulted from chromosomaltranslocation, for example, the Bcr/Abl fusion oncogene.

Further proteins include cyclin dependent kinases, c-myb, c-myc,proliferating cell nuclear antigen (PCNA), transforming growthfactor-beta (TGF-beta), and transcription factors nuclear factor kappaB(NF-.kappa.B), E2F, HER-2/neu, PKA, TGF-alpha, EGFR, TGF-beta, IGFIR,P12, MDM2, BRCA, Bcl-2, VEGF, MDR, ferritin, transferrin receptor, IRE,C-fos, HSP27, C-raf and metallothionein genes.

The siRNA employed in the present invention can be directed against thesynthesis of one or more proteins. Additionally or alternatively, therecan be more than one siRNA directed against a protein, e.g., duplicatesiRNA or siRNA that correspond to overlapping or non-overlapping targetsequences against the same target protein. Accordingly, in oneembodiment two, three, four or any plurality of siRNAs against the sametarget mRNA can be included in the nanoparticles of the invention.Additionally, several siRNAs directed against several proteins can beemployed. Alternatively, the siRNA can be directed against structural orregulatory RNA molecules that do not code for proteins.

In a preferred aspect of the invention, the target mRNA molecule of theinvention specifies the amino acid sequence of a protein associated witha pathological condition. For example, the protein may be apathogen-associated protein (e.g., a viral protein involved inimmunosuppression or immunoavoidance of the host, replication of thepathogen, transmission of the pathogen, or maintenance of theinfection), or a host protein which facilitates entry of the pathogeninto the host, drug metabolism by the pathogen or host, replication orintegration of the pathogen's genome, establishment or spread ofinfection in the host, or assembly of the next generation of pathogen.Alternatively, the protein may be a tumor-associated protein or anautoimmune disease-associated protein.

In one embodiment, the target mRNA molecule of the invention specifiesthe amino acid sequence of an endogenous protein (i.e. a protein presentin the genome of a cell or organism). In another embodiment, the targetmRNA molecule of the invention specifies the amino acid sequence of aheterologous protein expressed in a recombinant cell or a geneticallyaltered organism. In another embodiment, the target mRNA molecule of theinvention specifies the amino acid sequence of a protein encoded by atransgene (i.e., a gene construct inserted at an ectopic site in thegenome of the cell). In yet another embodiment, the target mRNA moleculeof the invention specifies the amino acid sequence of a protein encodedby a pathogen genome which is capable of infecting a cell or an organismfrom which the cell is derived.

By inhibiting the expression of such proteins, valuable informationregarding the function of said proteins and therapeutic benefits whichmay be obtained from said inhibition may be obtained.

In one embodiment, the nanoparticles of this invention comprises one ormore siRNA molecules to silence a PDGF beta gene, Erb-B gene, Src gene,CRK gene, GRB2 gene, RAS gene, MEKK gene, INK gene, RAF gene, Erkl/2gene, PCNA(p21) gene, MYB gene, JIJN gene, FOS gene, BCL-2 gene, CyclinD gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene,beta-catenin gene, c-MET gene, PKC gene, Skp2 gene, kinesin spindleprotein gene, Bcr-Abl gene, Stat3 gene, cSrc gene, PKC gene, Bax gene,Bcl-2 gene, EGFR gene, VEGF gene, myc gene, NFKB gene, STAT3 gene,survivin gene, Her2/Neu gene, topoisomerase I gene, PLK1 gene, proteinkinase 3 gene, CD31 gene, IGF-1 gene, topoisomerase II alpha gene,mutations in the p73 gene, mutations in the p21 (WAF 1/CIP 1) gene,mutations in the p27(KIP1) gene, mutations in the PPM1D gene, mutationsin the RAS gene, mutations in the caveolin I gene, mutations in the MIBI gene, mutations in the MTAI gene, mutations in the M68 gene, mutationsin tumor suppressor genes, mutations in the p53 tumor suppressor gene,mutations in the p53 family member DN-p63, mutations in the pRb tumorsuppressor gene, mutations in the APC1 tumor suppressor gene, mutationsin the BRCA1 tumor suppressor gene, mutations in the PTEN tumorsuppressor gene, mLL fusiongene, BCRIABL fusion gene, TEL/AML1 fusiongene, EWS/FLI1 fusion gene, TLS/FUS1 fusion gene, PAX3/FKHR fusion gene,AML1/ETO fusion gene, alpha v-integrin gene, Fit-i receptor gene,tubulin gene, Human Papilloma Virus gene, a gene required for HumanPapilloma Virus replication, Human Immunodeficiency Virus gene, a generequired for Human Immunodeficiency Virus replication, Hepatitis A Virusgene, a gene required for Hepatitis A Virus replication, Hepatitis BVirus gene, a gene required for Hepatitis B Virus replication, HepatitisC Virus gene, a gene required for Hepatitis C Virus replication,Hepatitis D Virus gene, a gene required for Hepatitis D Virusreplication, Hepatitis E Virus gene, a gene required for Hepatitis BVirus replication, Hepatitis F Virus gene, a gene required for HepatitisF Virus replication, Hepatitis G Virus gene, a gene required forHepatitis G Virus replication, Hepatitis H Virus gene, a gene requiredfor Hepatitis H Virus replication, Respiratory Syncytial Virus gene, agene that is required for Respiratory Syncytial Virus replication,Herpes Simplex Virus gene, a gene that is required for Herpes SimplexVirus replication, herpes Cytomegalovirus gene, a gene that is requiredfor herpes Cytomegalovirus replication, herpes Epstein Barr Virus gene,a gene that is required for herpes Epstein Barr Virus replication,Kaposi's Sarcoma-associated Herpes Virus gene, a gene that is requiredfor Kaposi's Sarcoma-associated Herpes Virus replication, JC Virus gene,human gene that is required for JC Virus replication, myxovirus gene, agene that is required for myxovirus gene replication, rhinovirus gene, agene that is required for rhinovirus replication, coronavirus gene, agene that is required for coronavirus replication, West Nile Virus gene,a gene that is required for West Nile Virus replication, St. LouisEncephalitis gene, a gene that is required for St. Louis Encephalitisreplication, Tick-borne encephalitis virus gene, a gene that is requiredfor Tick-borne encephalitis virus replication, Murray Valleyencephalitis virus gene, a gene that is required for Murray Valleyencephalitis virus replication, dengue virus gene, a gene that isrequired for dengue virus gene replication, Simian Virus 40 gene, a genethat is required for Simian Virus 40 replication, Human T CellLymphotropic Virus gene, a gene that is required for Human T CellLymphotropic Virus replication, Moloney-Murine Leukemia Virus gene, agene that is required for Moloney-Murine Leukemia Virus replication,encephalomyocarditis virus gene, a gene that is required forencephalomyocarditis virus replication, measles virus gene, a gene thatis required for measles virus replication, Vericella zoster virus gene,a gene that is required for Vericella zoster virus replication,adenovirus gene, a gene that is required for adenovirus replication,yellow fever virus gene, a gene that is required for yellow fever virusreplication, poliovirus gene, a gene that is required for poliovirusreplication, poxvirus gene, a gene that is required for poxvirusreplication, plasmodium gene, a gene that is required for plasmodiumgene replication, Mycobacterium ulcerans gene, a gene that is requiredfor Mycobacterium ulcerans replication, Mycobacterium tuberculosis gene,a gene that is required for Mycobacterium tuberculosis replication,Mycobacterium leprae gene, a gene that is required for Mycobacteriumleprae replication, Staphylococcus aureus gene, a gene that is requiredfor Staphylococcus aureus replication, Streptococcus pneumoniae gene, agene that is required for Streptococcus pneumoniae replication,Streptococcus pyogenes gene, a gene that is required for Streptococcuspyogenes replication, Chiamydia pneumoniae gene, a gene that is requiredfor Chiamydia pneumoniae replication, Mycoplasma pneumoniae gene, a genethat is required for Mycoplasma pneumoniae replication, an integringene, a selectin gene, complement system gene, chemokine gene, chemokinereceptor gene, GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4 gene, MIGgene, Pro-Platelet Basic Protein gene, MIP-11 gene, MIP-1J gene, RANTESgene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene,CMBKR3 gene, CMBKR5v, AIF-1 gene, 1-3 09 gene, a gene to a component ofan ion channel, a gene to a neurotransmitter receptor, a gene to aneurotransmitter ligand, amyloid-family gene, presenilin gene, HD gene,DRPLA gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene, SCA7 gene,SCA8 gene, allele gene found in LOH cells, or one allele gene of apolymorphic gene. Examples of relevant siRNA molecules to silence genesand methods of making siRNA molecules can be found from commercialsources such as Dharmacon or from the following patent applications:US2005017667, WO2006066158, WO2006078278, U.S. Pat. No. 7,056,704, U.S.Pat. No. 7,078,196, U.S. Pat. No. 5,898,031, U.S. Pat. No. 6,107,094, EP1144623, and EU 1144623, all of which are incorporated herein byreference in their entireties. While a number of specific gene silencingtargets are listed, this list is merely illustrative and other siRNAmolecules could also be used with the nanoparticles of this invention.

In one embodiment, the nanoparticles of this invention comprise an siRNAmolecule having RNAi activity against an RNA, wherein the siRNA moleculecomprises a sequence complementary to any RNA having coding ornon-encoding sequence, such as those sequences referred to by GenBankAccession Nos. described in Table V of PCT/US03/05028 (International PCTPublication No. WO 03/4654) or otherwise known in the art.

In one embodiment, the nanoparticles of this invention comprise an siRNAmolecule which silences the vascular endothelial growth factor gene. Inanother embodiment, the nanoparticles of this invention comprise ansiRNA molecule which silences the vascular endothelial growth factorreceptor gene.

In another embodiment, the nanoparticles of this invention comprise ansiRNA molecule, wherein the sequence of the siRNA molecule iscomplementary to tumor-related targets, including, but not limited to,hypoxia-inducible factor-1 (HIF-1), which is found in human metastaticprostate PC3-M cancer cells (Mol Carcinog. 2008 Jan. 31 [Epub ahead ofprint]); the HIF-1 downstream target gene (Mol Carcinog. 2008 Jan. 31[Epub ahead of print]), mitogen-activated protein kinases (MAPKs),hepatocyte growth factor (HGF), interleukin 12p70 (IL12),glucocorticoid-induced tumor necrosis factor receptor (GITR),intercellular adhesion molecule 1 (ICAM-1), neurotrophin-3 (NT-3),interleukin 17 (IL17), interleukin 18 binding protein a (IL18 Bpa) andepithelial-neutrophil activating peptide (ENA78) (see, e.g., “Cytokineprofiling of prostatic fluid from cancerous prostate glands identifiescytokines associated with extent of tumor and inflammation”, TheProstate Early view Published Online: 24 Mar. 2008); PSMA (see, e.g.,“Cell-Surface labeling and internalization by a fluorescent inhibitor ofprostate-specific membrane antigen” The Prostate Early view PublishedOnline: 24 Mar. 2008); Androgen receptor (AR), keratin, epithelialmembrane antigen, EGF receptor, and E cadherin (see, e.g.,“Characterization of PacMetUT1, a recently isolated human prostatecancer cell line”); peroxisomes proliferators-activated receptor γ(PPARγ; see e.g., The Prostate Volume 68, Issue 6, Date: 1 May 2008,Pages: 588-598); the receptor for advanced glycation end products (RAGE)and the advanced glycation end products (AGE), (see, e.g., “V domain ofRAGE interacts with AGEs on prostate carcinoma cells” The Prostate Earlyview Published Online: 26 Feb. 2008); the receptor tyrosine kinaseerb-B2 (Her2/neu), hepatocyte growth factor receptor (Met), transforminggrowth factor-beta 1 receptor (TGFβR1), nuclear factor kappa B (NFκB),Jagged-1, Sonic hedgehog (Shh), Matrix metalloproteinases (MMPs, esp.MMP-7), Endothelin receptor type A (ET_(A)), Endothelin-1 (ET-1),Nuclear receptor subfamily 3, group C, member 1 (NR3C1), Nuclearreceptor co-activator 1 (NCOA1), NCOA2, NCOA3, E1A binding protein p300(EP300), CREB binding protein (CREBBP), Cyclin G associated kinase(GAK), Gelsolin(GSN), Aldo-keto reductase family 1, member C1 (AKR1C1),AKR1C2, AKR1C3, Neurotensin(NTS), Enolase 2(ENO2), Chromogranin B (CHGB,secretogranin 1), Secretagogin (SCGN, or EF-hand calcium bindingprotein), Dopa decarboxylase (DDC, or aromatic L-amino aciddecarboxylase), steroid receptor co-activator-1 (SRC-1), SRC-2 (a.k.a.TIF2), SRC-3 (a.k.a. AIB-1) (see, e.g., “Longitudinal analysis ofandrogen deprivation of prostate cancer cells identifies pathways toandrogen independence” The Prostate Early view Published Online: 26 Feb.2008); estrogen receptors (ERα, ERβ or GPR30) (see, e.g., The ProstateVolume 68, Issue 5, Pages 508-516); the melanoma cell adhesion molecule(MCAM) (see, e.g., The Prostate Volume 68, Issue 4, Pages 418-426;angiogenic factors (such as vascular endothelial growth factor (VEGF)and erythropoietin), glucose transporters (such as GLUT1),BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) (see, e.g., TheProstate Volume 68, Issue 3, Pages 336-343); types 1 and 2 5α-reductase(see, e.g., The Journal of Urology Volume 179, Issue 4, Pages1235-1242); ERG and ETV1, prostate-specific antigen (PSA),prostate-specific membrane antigen (PSMA), prostate stem cell antigen(PSCA), α-Methylacyl coenzyme A racemase (AMACR), PCA3^(DD3),glutathione-S-transferase, pi 1 (GSTP1), p16, ADP-ribosylation factor(ARF), O-6-methylguanine-DNA methyltransferase (MGMT), human telomerasereverse transcriptase (hTERT), early prostate cancer antigen (EPCA),human kallikrein 2 (HK2) and hepsin (see, e.g., The Journal of UrologyVolume 178, Issue 6, Pages 2252-2259); bromodomain containing 2 (BRD2),eukaryotic translation initiation factor 4 gamma, 1 (eIF4G1), ribosomalprotein L13a (RPL13a), and ribosomal protein L22 (RPL22) (see, e.g., NEngl J Med 353 (2005), p. 1224); HER2/neu, Derlin-1, ERBB2, AKT,cyclooxygenase-2 (COX-2), PSMD3, CRKRS, PERLD1, and C17ORF37, PPP4C,PARN, ATP6V0C, C16orf14, GBL, HAGH, ITFG3, MGC13114, MRPS34, NDUFB10,NMRAL1, NTHL1, NUBP2, POLR3K, RNPS1, STUB1, TBL3, and USP7. All of thereferences described herein are incorporated herein by reference intheir entireties.

Thus, in one embodiment, the invention comprises a nanoparticlecomprising a targeting moiety (e.g., PSMA ligand), a biodegradablepolymer, a stealth polymer, and an siRNA molecule wherein the siRNAmolecule is encapsulated within a reverse micelle. In one embodiment,the invention comprises a nanoparticle comprising a targeting moiety(e.g., PSMA ligand), a biodegradable polymer, a stealth component, andan siRNA molecule that silences the vascular endothelial growth factorgene, wherein the siRNA molecule is encapsulated within a reversemicelle. In one embodiment, the invention comprises a nanoparticlecomprising a targeting moiety (e.g., PSMA ligand), a biodegradablepolymer, a stealth component, and an siRNA molecule that silences thevascular endothelial growth factor receptor gene, wherein the siRNAmolecule is encapsulated within a reverse micelle. In anotherembodiment, the invention comprises a nanoparticle comprising atargeting moiety (e.g., PSMA ligand), PLGA, polyethylene glycol, and ansiRNA molecule, wherein the siRNA molecule is encapsulated within areverse micelle. In one embodiment, the invention comprises ananoparticle comprising a targeting moiety (e.g., PSMA ligand), abiodegradable polymer, a stealth component, and an siRNA moleculewherein the nanoparticle can selectively accumulate in the prostate orin the vascular endothelial tissue surrounding a cancer, wherein thesiRNA molecule is encapsulated within a reverse micelle. In oneembodiment, the invention comprises a nanoparticle comprising atargeting moiety (e.g., PSMA ligand), a biodegradable polymer, a stealthcomponent, and an siRNA molecule wherein the nanoparticle canselectively accumulate in the prostate or in the vascular endothelialtissue surrounding a cancer and wherein the nanoparticle can beendocytosed by a PSMA expressing cell, wherein the siRNA molecule isencapsulated within a reverse micelle.

In one embodiment, the invention comprises a nanoparticle comprising oneor a plurality of the following: natural or unnatural siRNAs, shRNAs,microRNAs, ribozymes, DNA enzymes, aptamers, antisense oligonucleotides,randomized oligonucleotides, DNA plasmids, etc. In another embodiment,the encapsulated nucleic acids include, but are not limited to,noncoding RNAs, for example, rRNA, snRNA, snoRNA and tRNA, etc. Incertain embodiments, the encapsulated nucleic acids are smallinterfering RNAs or short interfering RNAs (siRNAs), referring to an RNA(or RNA analog) comprising between about 10-50 nucleotides (ornucleotide analogs) which is capable of directing or mediating RNAinterference. In some preferred embodiments, the targets of RNAinterference include, for example, the target genes exemplified in thisinvention.

In other embodiments, the encapsulated nucleic acids are natural orunnatural short hairpin RNAs (shRNAs) capable of inducing RNAinterference with a stable knockdown, by expressing mimics of micro RNAs(miRNAs) from RNA polymerase II or III promoters (e.g., as described in“Unlocking the potential of the human genome with RNA interference”Nature 431, 371-378). In one preferred embodiment, shRNAs typically havestems ranging from 19 to 29 nucleotides in length, and with variousdegrees of structural similarity to nature miRNAs. In another preferredembodiment, shRNAs are expressed under Pol III promoter withoutimmunostimulatory effects as siRNAs do (e.g., as described in “Stableexpression of shRNAs in human CD34+ progenitor cells can avoid inductionof interferon responses to siRNAs in vitro” Nature Biotechnology 24,566-571). In one embodiment, the shRNA is expressed in commerciallyavailable vectors, e.g., from OriGene Technologies, Inc., which arebased on a HuSH pRS plasmid vector which contains both 5 and 3 LTRs ofMoloney murine leukemia virus (MMLV) that flank the puromycin marker andthe U6-shRNA expression cassette. In one preferred embodiment, the shRNAexpression cassette consists of a 21 bp target gene specific sequence, a10 bp loop, another 21 bp reverse complementary sequence, and atermination sequence to terminate the transcription by RNA Pol III, allunder a human U6 promoter. In one preferred embodiment, the 21 bpgene-specific sequence is sequence-verified to ensure its match to thetarget gene, which includes, for example, the target genes of siRNAsdescribed in this invention.

In some embodiments, the encapsulated nucleic acids are natural orunnatural miRNAs, which are isolated from an organism or synthesized bystandard techniques, respectively. In one embodiment, a miRNA is asingle-stranded RNA of typically 19-25 nucleotides length, also referredto as a mature miRNA. In one preferred embodiment, the miRNAs act aspost-transcriptional regulators of gene expression during cancer ortumor formation by base-pairing with their target mRNAs. In oneembodiment, the miRNAs are encoded in a DNA plasmid and transfected intothe cell. In one preferred embodiment, the target genes of the miRNAsinclude the target genes of siRNA described in this invention, forexample, cytokines, proto-oncogenes, oncogenes (e.g., myc, ras, etc.)and other disease markers.

In some embodiments, the encapsulated nucleic acids are natural orunnatural ribozymes. In one embodiment, ribozyme molecules are designedto catalytically cleave target mRNA transcripts (e.g., as described inPCT International Publication WO90/11364; Sarver et al., 1990, Science247: 1222-1225 and U.S. Pat. No. 5,093,246, which are incorporatedherein in their entireties). In one preferred embodiment, hammerheadribozymes are used to destroy target mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The construction and production of hammerhead ribozymes is wellknown in the art and is described more fully in Haseloff and Gerlach,1988, Nature, 334:585-591. In other embodiments, the ribozymes alsoinclude RNA endoribonucleases (“Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena thermophia (known as the IVS, orL-19 IVS RNA) (e.g. as described in International patent application No.WO88/04300).

In some embodiments, the encapsulated nucleic acids are natural orunnatural DNA enzymes. In one preferred embodiments, the DNA enzymesinclude two basic types, as identified by Santoro and Joyce (see, forexample, U.S. Pat. No. 6,110,462). In some embodiments, the DNA enzymesare designed to specifically recognize and cleave a target nucleic acid.The target nucleic acid can be identified by one of skill in the art,using the same approach as outlined for siRNA. In one preferredembodiment, the unique or substantially sequence is a G/C rich ofapproximately 18 to 22 nucleotides.

In some embodiments, the encapsulated nucleic acids are natural orunnatural aptamers. In one preferred embodiment, the aptamers arenucleic acid molecules specifically binding to molecules throughinteractions other than classic Watson-Crick base pairing. In oneembodiment, the aptamers are generated for proteins including growthfactors, transcription factors, enzymes, immunoglobulins, and receptors(see, for example, U.S. patent Ser. No. 10/980,211). In one preferredembodiment, the aptamers are 10-15 kDa in size (30-45 nucleotides), bindtheir targets with sub-nanomolar affinity, and discriminate againstclosely related targets (e.g. proteins in the same gene family). Thetarget selection for aptamers is well known in the art and includes, forexample, the target genes of siRNAs described in this invention.

In some embodiments, the encapsulated nucleic acids are natural orunnatural antisense oligonucleotides. In one preferred embodiment, theantisense oligonucleotide is an isolated, synthetic, substantially pure,or recombinant polynucleotide having a sequence that is at least about10 nucleotides in length to at least about 100 nucleotides in length.This polynucleotide comprises a sequence exactly complementary oridentical to a contiguous sequence of a nucleic acid encoding anendogenous protein, or a mutated protein in the target cell. The targetof the antisense oligonucleotides includes, for example, cytokines,proto-oncogenes, oncogenes (e.g., myc, ras, etc.) and other cancer ortumor-related genes, including the target genes of siRNA described inthis invention. (see, for example, U.S. patent Ser. No. 10/637,443). Thedesigning for antisense oligonucleotides is well known in the art.

In some embodiments, the encapsulated nucleic acids are randomizedoligonucleotides. In one preferred embodiment, the randomizedoligonucleotides are designed to consist essentially of one or moreoligonucleotides capable of binding a DNA-binding protein or RNA primersin the target cell, thereby treating the cancer or tumor. The designingfor randomized oligonucleotides is well known in the art and described,for example, in U.S. patent application Ser. No. 10/613,390).

In some embodiments, the encapsulated nucleic acids are DNA plasmids. Inone preferred embodiments, the DNA plasmids are vectors to transportanother nucleic acid to which it has been linked, achieveextra-chromosomal replication and/or expression of nucleic acid to whichthey are linked in a host cell (e.g., a cell targeted by targetedparticles of the present invention). In some embodiments, the DNAplasmids can achieve integration into the genome of the host cell. Insome embodiments, the DNA plasmids are used to direct protein and/or RNAexpression. In one embodiment, the protein and/or RNA to be expressed isnot normally expressed by the cell. In another embodiment, the proteinand/or RNA to be expressed is normally expressed by the cell, but atlower levels than it is expressed when the plasmid has not beendelivered to the cell. In one preferred embodiment, the DNA plasmids aredesigned to express siRNAs, shRNAs, miRNAs, ribozymes, DNA enzymes,apatamers, antisense oligonucleotides or randomized oligonucleotidesdescribed in the present invention.

In another embodiment, the nucleic acid (e.g. siRNA, shRNA, miRNA, etc.)that is incorporated into the nanoparticle of the invention are thosethat treat prostate cancer, such as those disclosed in U.S. patentapplication Ser. No. 11/021,159 (siRNA sequence is complementary togaaggccagu uguauggac (SEQ ID NO: 13)), and U.S. application Ser. No.11/349,473 Both of these references are incorporated herein by referencein their entirety.

In another embodiment, the therapeutic agents of the nanoparticles ofthe invention include RNAs that can be used to treat cancer, such asanti-sense mRNAs and microRNAs. Examples of microRNAs that can be usedas therapeutic agents for the treatment of cancer include thosedisclosed in Nature 435 (7043): 828-833; Nature 435 (7043): 839-843; andNature 435 (7043): 834-838, all of which are incorporated herein byreference in their entireties.

Once the inventive conjugates have been prepared, they may be combinedwith pharmaceutical acceptable carriers to form a pharmaceuticalcomposition, according to another aspect of the invention. As would beappreciated by one of skill in this art, the carriers may be chosenbased on the route of administration as described below, the location ofthe target issue, the drug being delivered, the time course of deliveryof the drug, etc.

Methods of Treatment

In some embodiments, targeted particles in accordance with the presentinvention may be used to treat, alleviate, ameliorate, relieve, delayonset of, inhibit progression of, reduce severity of, and/or reduceincidence of one or more symptoms or features of a disease, disorder,and/or condition. In some embodiments, the targeted nanoparticles of theinvention can be used to treat cancer, e.g., prostate or breast cancer,and/or cancer cells, e.g., prostate or breast cancer cells in a subjectin need thereof. In other embodiments, the targeted nanoparticles of theinvention can be used to treat atherosclerotic plaques, restenosis, andatherosclerosis in a subject in need thereof.

The term “subject” is intended to include organisms, e.g., prokaryotesand eukaryotes, which are capable of suffering from or afflicted with adisease or disorder. Examples of subjects include mammals, e.g., humans,dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, andtransgenic non-human animals. In certain embodiments, the subject is ahuman, e.g., a human suffering from, at risk of suffering from, orpotentially capable of suffering from a disease or disorder.

The term “cancer” includes pre-malignant as well as malignant cancers.Cancers include, but are not limited to, prostate, gastric cancer,colorectal cancer, skin cancer, e.g., melanomas or basal cellcarcinomas, lung cancer, cancers of the head and neck, bronchus cancer,pancreatic cancer, urinary bladder cancer, brain or central nervoussystem cancer, peripheral nervous system cancer, esophageal cancer,cancer of the oral cavity or pharynx, liver cancer, kidney cancer,testicular cancer, biliary tract cancer, small bowel or appendix cancer,salivary gland cancer, thyroid gland cancer, adrenal gland cancer,osteosarcoma, chondrosarcoma, cancer of hematological tissues, and thelike. “Cancer cells” can be in the form of a tumor, exist alone within asubject (e.g., leukemia cells), or be cell lines derived from a cancer.

Cancer can be associated with a variety of physical symptoms. Symptomsof cancer generally depend on the type and location of the tumor. Forexample, lung cancer can cause coughing, shortness of breath, and chestpain, while colon cancer often causes diarrhea, constipation, and bloodin the stool. However, to give but a few examples, the followingsymptoms are often generally associated with many cancers: fever,chills, night sweats, cough, dyspnea, weight loss, loss of appetite,anorexia, nausea, vomiting, diarrhea, anemia, jaundice, hepatomegaly,hemoptysis, fatigue, malaise, cognitive dysfunction, depression,hormonal disturbances, neutropenia, pain, non-healing sores, enlargedlymph nodes, peripheral neuropathy, and sexual dysfunction.

In one aspect of the invention, a method for the treatment of cancer(e.g. breast or prostate cancer) is provided. In some embodiments, thetreatment of cancer comprises administering a therapeutically effectiveamount of inventive targeted particles to a subject in need thereof, insuch amounts and for such time as is necessary to achieve the desiredresult. In certain embodiments of the present invention a“therapeutically effective amount” of an inventive targeted particle isthat amount effective for treating, alleviating, ameliorating,relieving, delaying onset of, inhibiting progression of, reducingseverity of, and/or reducing incidence of one or more symptoms orfeatures of cancer.

In one aspect of the invention, a method for administering inventivecompositions to a subject suffering from cancer (e.g. breast or prostatecancer) is provided. In some embodiments, particles to a subject in suchamounts and for such time as is necessary to achieve the desired result(i.e., treatment of cancer). In certain embodiments of the presentinvention a “therapeutically effective amount” of an inventive targetedparticle is that amount effective for treating, alleviating,ameliorating, relieving, delaying onset of, inhibiting progression of,reducing severity of, and/or reducing incidence of one or more symptomsor features of cancer.

In other embodiments, the nanoparticles of the present invention can beused to inhibit the growth of cancer cells, e.g., prostate or breastcancer cells. As used herein, the term “inhibits growth of cancer cells”or “inhibiting growth of cancer cells” refers to any slowing of the rateof cancer cell proliferation and/or migration, arrest of cancer cellproliferation and/or migration, or killing of cancer cells, such thatthe rate of cancer cell growth is reduced in comparison with theobserved or predicted rate of growth of an untreated control cancercell. The term “inhibits growth” can also refer to a reduction in sizeor disappearance of a cancer cell or tumor, as well as to a reduction inits metastatic potential. Preferably, such an inhibition at the cellularlevel may reduce the size, deter the growth, reduce the aggressiveness,or prevent or inhibit metastasis of a cancer in a patient. Those skilledin the art can readily determine, by any of a variety of suitableindicia, whether cancer cell growth is inhibited.

Inhibition of cancer cell growth may be evidenced, for example, byarrest of cancer cells in a particular phase of the cell cycle, e.g.,arrest at the G2/M phase of the cell cycle. Inhibition of cancer cellgrowth can also be evidenced by direct or indirect measurement of cancercell or tumor size. In human cancer patients, such measurementsgenerally are made using well known imaging methods such as magneticresonance imaging, computerized axial tomography and X-rays. Cancer cellgrowth can also be determined indirectly, such as by determining thelevels of circulating carcinoembryonic antigen, prostate specificantigen or other cancer-specific antigens that are correlated withcancer cell growth. Inhibition of cancer growth is also generallycorrelated with prolonged survival and/or increased health andwell-being of the subject.

The present invention is also directed, in part, to the discovery that acollagen IV alpha-2 chain related polypeptide can act as a receptor forthe CREKA (SEQ ID NO: 4) tumor targeting peptide. Collagens are a majorcomponent of the extracellular matrix (ECM), an interconnected molecularnetwork providing mechanical support for cells and tissues andregulating biochemical and cellular processes such as adhesion,migration, gene expression and differentiation (see, e.g., U.S. PatentApplication No. 2005/0048063, which is incorporated herein by referencein its entirety). In higher animals, at least 19 distinct collagen typesdiffering in their higher order structures and functions have beenidentified based on the presence of the characteristic collagentriple-helix structure. The collagens are sometimes categorized into thefibrillar and nonfibrillar collagens. The fibrillar (interstitial)collagens include types I, II, III, V and XI, while the nonfibrillarcollagens include types IV, VI, IX, X, XI, XII, XIV and XIII.

Targeting moieties useful in the invention include those whichselectively target tumor vasculature and selectively bind non-helicalcollagen. Targeting moieties include those which selectively target totumor vasculature and selectively bind collagen IV, and those whichselectively target tumor vasculature and selectively bind denaturedcollagen IV in preference to native collagen IV. Such moieties include,but are not limited to, AKERC (SEQ ID NO: 3), CREKA (SEQ ID NO: 4),ARYLQKLN (SEQ ID NO: 5) or AXYLZZLN (SEQ ID NO: 6), wherein X and Z arevariable amino acids. As such, the nanoparticles of the invention can beused for the treatment of vulnerable plaques in a subject in needthereof.

In a preferred embodiment, the nanoparticles of the invention can bedelivered to or near a vulnerable plaque, particularly when thetargeting moiety is a peptide that targets the basement membrane of ablood vessel (e.g., CREKA), using a medical device such as a needlecatheter, drug eluding stent or stent graft. Such devices are well knownin the art, and are described, for example, in U.S. Pat. No. 7,008,411,which is incorporated herein by reference in its entirety. In oneembodiment, a drug eluting stent and/or needle catheter may be implantedat the region of vessel occlusion that may be upstream from a vulnerableplaque region. A medical device, such as a drug eluting stent, needlecatheter, or stent graft may be used to treat the occlusiveatherosclerosis (i.e., non-vulnerable plaque) while releasing thenanoparticle of the invention to treat a vulnerable plaque region distalor downstream to the occlusive plaque. The nanoparticle may be releasedslowly over time.

The nanoparticles of the invention can also be delivered to a subject inneed thereof using the Genie™ balloon catheter available from Acrostak(http://www.acrostak.com/genie_en.htm). The nanoparticles of theinvention can also be delivered to a subject in need thereof usingdelivery devices that have been developed for endovascular local genetransfer such as passive diffusion devices (e.g., double-occlusionballoon, spiral balloon), pressure-driven diffusion devices (e.g.,microporous balloon, balloon-in-balloon devices, double-layer channeledperfusion balloon devices, infusion-sleeve catheters, hydrogel-coatedballoons), and mechanically or electrically enhanced devices (e.g.,needle injection catheter, iontophoretic electric current-enhancedballoons, stent-based system), or any other delivery system disclosed inRadiology 2003; 228:36-49, or Int J Nanomedicine 2007; 2(2):143-61,which are incorporated herein by reference in their entireties.

Pharmaceutical Compositions

As used herein, the term “pharmaceutically acceptable carrier” means anon-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. Remington'sPharmaceutical Sciences. Ed. by Gennaro, Mack Publishing, Easton, Pa.,1995 discloses various carriers used in formulating pharmaceuticalcompositions and known techniques for the preparation thereof. Someexamples of materials which can serve as pharmaceutically acceptablecarriers include, but are not limited to, sugars such as lactose,glucose, and sucrose; starches such as com starch and potato starch;cellulose and its derivatives such as sodium carboxymethyl cellulose,ethyl cellulose, and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients such as cocoa butter and suppository waxes;oils such as peanut oil, cottonseed oil; safflower oil; sesame oil;olive oil; corn oil and soybean oil; glycols such as propylene glycol;esters such as ethyl oleate and ethyl laurate; agar; detergents such asTWEEN™ 80; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator. If filtration or otherterminal sterilization methods are not feasible, the formulations can bemanufactured under aseptic conditions.

The pharmaceutical compositions of this invention can be administered toa patient by any means known in the art including oral and parenteralroutes. According to such embodiments, inventive compositions may beadministered by injection (e.g., intravenous, subcutaneous orintramuscular, intraperitoneal injection), rectally, vaginally,topically (as by powders, creams, ointments, or drops), or by inhalation(as by sprays).

In a particular embodiment, the nanoparticles of the present inventionare administered to a subject in need thereof systemically, e.g., by IVinfusion or injection.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P., and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Inone embodiment, the inventive conjugate is suspended in a carrier fluidcomprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) TWEEN™80. The injectable formulations can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Compositions for rectal or vaginal administration may be suppositorieswhich can be prepared by mixing the inventive conjugate with suitablenon-irritating excipients or carriers such as cocoa butter, polyethyleneglycol, or a suppository wax which are solid at ambient temperature butliquid at body temperature and therefore melt in the rectum or vaginalcavity and release the inventive conjugate.

Dosage forms for topical or transdermal administration of an inventivepharmaceutical composition include ointments, pastes, creams, lotions,gels, powders, solutions, sprays, inhalants, or patches. The inventiveconjugate is admixed under sterile conditions with a pharmaceuticallyacceptable carrier and any needed preservatives or buffers as may berequired. Ophthalmic formulations, ear drops, and eye drops are alsocontemplated as being within the scope of this invention. The ointments,pastes, creams, and gels may contain, in addition to the inventiveconjugates of this invention, excipients such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, andzinc oxide, or mixtures thereof. Transdermal patches have the addedadvantage of providing controlled delivery of a compound to the body.Such dosage forms can be made by dissolving or dispensing the inventiveconjugates in a proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the inventive conjugates in a polymer matrix or gel.

Powders and sprays can contain, in addition to the inventive conjugatesof this invention, excipients such as lactose, talc, silicic acid,aluminum hydroxide, calcium silicates, and polyamide powder, or mixturesthereof. Sprays can additionally contain customary propellants such aschlorofluorohydrocarbons.

When administered orally, the inventive nanoparticles can be, but arenot necessarily, encapsulated. A variety of suitable encapsulationsystems are known in the art (“Microcapsules and Nanoparticles inMedicine and Pharmacy,” Edited by Doubrow, M., CRC Press, Boca Raton,1992; Mathiowitz and Langer J. Control. Release 5:13, 1987; Mathiowitzet al. Reactive Polymers 6:275, 1987; Mathiowitz et al. J. Appl. PolymerSci. 35:755, 1988; Langer Ace. Chem. Res. 33:94, 2000; Langer J.Control. Release 62:7, 1999; Uhrich et al. Chem. Rev. 99:3181, 1999;Zhou et al. J. Control. Release 75:27, 2001; and Hanes et al. Pharm.Biotechnol. 6:389, 1995). The inventive conjugates may be encapsulatedwithin biodegradable polymeric microspheres or liposomes. Examples ofnatural and synthetic polymers useful in the preparation ofbiodegradable microspheres include carbohydrates such as alginate,cellulose, polyhydroxyalkanoates, polyamides, polyphosphazenes,polypropylfumarates, polyethers, polyacetals, polycyanoacrylates,biodegradable polyurethanes, polycarbonates, polyanhydrides,polyhydroxyacids, poly(ortho esters), and other biodegradablepolyesters. Examples of lipids useful in liposome production includephosphatidyl compounds, such as phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides.

Pharmaceutical compositions for oral administration can be liquid orsolid. Liquid dosage forms suitable for oral administration of inventivecompositions include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and elixirs. In additionto an encapsulated or unencapsulated conjugate, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants, wetting agents, emulsifying andsuspending agents, sweetening, flavoring, and perfuming agents. As usedherein, the term “adjuvant” refers to any compound which is anonspecific modulator of the immune response. In certain embodiments,the adjuvant stimulates the immune response. Any adjuvant may be used inaccordance with the present invention. A large number of adjuvantcompounds is known in the art (Allison Dev. Biol. Stand. 92:3-11, 1998;Unkeless et al. Annu. Rev. Immunol. 6:251-281, 1998; and Phillips et al.Vaccine 10:151-158, 1992).

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, theencapsulated or unencapsulated conjugate is mixed with at least oneinert, pharmaceutically acceptable excipient or carrier such as sodiumcitrate or dicalcium phosphate and/or (a) fillers or extenders such asstarches, lactose, sucrose, glucose, mannitol, and silicic acid, (b)binders such as, for example, carboxymethylcellulose, alginates,gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectantssuch as glycerol, (d) disintegrating agents such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate, (e) solution retarding agents such as paraffin,(f) absorption accelerators such as quaternary ammonium compounds, (g)wetting agents such as, for example, cetyl alcohol and glycerolmonostearate, (h) absorbents such as kaolin and bentonite clay, and (i)lubricants such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof. Inthe case of capsules, tablets, and pills, the dosage form may alsocomprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart.

It will be appreciated that the exact dosage of the targeted reversemicelle particle is chosen by the individual physician in view of thepatient to be treated, in general, dosage and administration areadjusted to provide an effective amount of the targeted particle to thepatient being treated. As used herein, the “effective amount” of atargeted particle refers to the amount necessary to elicit the desiredbiological response. As will be appreciated by those of ordinary skillin this art, the effective amount of targeted particle may varydepending on such factors as the desired biological endpoint, the drugto be delivered, the target tissue, the route of administration, etc.For example, the effective amount of targeted particle containing ananti-cancer drug might be the amount that results in a reduction intumor size by a desired amount over a desired period of time. Additionalfactors which may be taken into account include the severity of thedisease state; age, weight and gender of the patient being treated;diet, time and frequency of administration; drug combinations; reactionsensitivities; and tolerance/response to therapy.

The nanoparticles of the invention may be formulated in dosage unit formfor ease of administration and uniformity of dosage. The expression“dosage unit form” as used herein refers to a physically discrete unitof nanoparticle appropriate for the patient to be treated. It will beunderstood, however, that the total daily usage of the compositions ofthe present invention will be decided by the attending physician withinthe scope of sound medical judgment. For any nanoparticle, thetherapeutically effective dose can be estimated initially either in cellculture assays or in animal models, usually mice, rabbits, dogs, orpigs. The animal model is also used to achieve a desirable concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.Therapeutic efficacy and toxicity of nanoparticles can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose is therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose is lethal to 50% of the population). Thedose ratio of toxic to therapeutic effects is the therapeutic index, andit can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceutical compositionswhich exhibit large therapeutic indices may be useful in someembodiments. The data obtained from cell culture assays and animalstudies can be used in formulating a range of dosage for human use.

The present invention also provides any of the above-mentionedcompositions in kits, optionally with instructions for administering anyof the compositions described herein by any suitable technique aspreviously described, for example, orally, intravenously, pump orimplantable delivery device, or via another known route of drugdelivery. “Instructions” can define a component of promotion, andtypically involve written instructions on or associated with packagingof compositions of the invention. Instructions also can include any oralor electronic instructions provided in any manner. The “kit” typicallydefines a package including any one or a combination of the compositionsof the invention and the instructions, but can also include thecomposition of the invention and instructions of any form that areprovided in connection with the composition in a manner such that aclinical professional will clearly recognize that the instructions areto be associated with the specific composition.

The kits described herein may also contain one or more containers, whichmay contain the inventive composition and other ingredients aspreviously described. The kits also may contain instructions for mixing,diluting, and/or administrating the compositions of the invention insome cases. The kits also can include other containers with one or moresolvents, surfactants, preservative and/or diluents (e.g., normal saline(0.9% NaCl), or 5% dextrose) as well as containers for mixing, dilutingor administering the components in a sample or to a subject in need ofsuch treatment.

The compositions of the kit may be provided as any suitable form, forexample, as liquid solutions or as dried powders. When the compositionprovided is a dry powder, the composition may be reconstituted by theaddition of a suitable solvent, which may also be provided. Inembodiments where liquid forms of the composition are used, the liquidform may be concentrated or ready to use. The solvent will depend on thenanoparticle and the mode of use or administration. Suitable solventsfor drug compositions are well known, for example as previouslydescribed, and are available in the literature. The solvent will dependon the nanoparticle and the mode of use or administration.

The invention also involves, in another aspect, promotion of theadministration of any of the nanoparticles described herein. In someembodiments, one or more compositions of the invention are promoted forthe prevention or treatment of various diseases such as those describedherein via administration of any one of the compositions of the presentinvention. As used herein, “promoted” includes all methods of doingbusiness including methods of education, hospital and other clinicalinstruction, pharmaceutical industry activity including pharmaceuticalsales, and any advertising or other promotional activity includingwritten, oral and electronic communication of any form, associated withcompositions of the invention.

EXAMPLES

The invention is further illustrated by the following examples. Theexamples should not be construed as further limiting.

Preparation of Polymer-Encapsulated Reverse Micelles

In this example, an anti-polo-like kinase 1 siRNA (anti-PLK1) can beresuspended in water at 1 mg/mL. Fifty microliters of this solution canbe mixed with 1 mL of a tetrahydrofuran solution containing lecithin (acommercially available plant-derived mixture of glycolipids,triglycerides, and phospholipids) at 0.1 mg/mL to formlecithin-anti-PLK1 reverse micelles. These reverse micelles can be addedto a 1 mL solution containing 5 mg/mL of poly(D,L-lactic-co-glycolicacid)-block-poly(ethylene glycoi-block-A10 anti-PSMA aptamer)(PLGA-PEG-Apt) triblock copolymer dissolved in tetrahydrofuran. Thismixture of lecithin-anti-PLK1 reverse micelles and PLGA-PEG-Apt can beadded dropwise to 3 mL of a DNase/RNase free water solution to formanti-PLK1-containing nanoparticles that present anti-PSMA aptamers onthe surface by the nanoprecipitation method. This procedure would yieldnanoparticles targeting an anti-PLK1 siRNA to PSMA-expressing cells,such as LNCaP prostate adenocarcinomas, to produce selective cellkilling.

In a second embodiment, DNA oligonucleotides of known sequence may beencapsulated efficiently into nanoparticles of different physicochemicalproperties, such that each distinct nanoparticle formulation contains aknown and unique oligonucleotide. The nanoparticles of differentphysicochemical characteristics may then be screened in parallel eitherin vitro or in vivo for preferential cell uptake or tumor targeting,respectively, by collecting the cells or the tumor and assaying for thetotal content of each oligonucleotide by ELISA, sandwich capture assay,or quantitative RT-PCR. Nanoparticles of biodegradable polymers may thenbe selected for a particular application by correlating the levels ofoligonucleotide present with the levels of nanoparticles present in thetargeted location.

Encapsulation efficiency is determined by taking a known amount of DNA,encapsulating it into a nanoparticle, removing any unencapsulated DNA byfiltration, lysing the nanoparticle, then detecting the amount of DNAthat was encapsulated by measuring its absorbance of light at 260 nm.The encapsulation efficiency is calculated by taking the amount of DNAthat was encapsulated, then dividing it by the original amount of DNA.Stated alternatively, it is the fraction of initial DNA that issuccessfully encapsulated.

Zeta potential is determined by Quasi-elastic laser light scatteringwith a ZetaPALS dynamic light scattering detector (BrookhavenInstruments Corporation, Holtsville, N.Y.; 15 mW laser, incidentbeam=676 nm).

FIG. 1 demonstrates the effect of amphipathic lipid concentration onencapsulation particle size.

FIG. 2 demonstrates the effect of amphipathic lipid concentration onparticle zeta potential. PLGA-PEG and DDAB (dimethyldiotadecylammoniumbromide), a cationic lipid, were used as the polymer and lipid in thereverse micelle nanoprecipitation protocol. As the ratio of lipid:DNA isincreased, one can see that there is a point between the ratios of 35:1and 351:1 where the cationic lipid finds its way to the surface of theparticle in sufficient quantities to yield an overall net positivesurface charge. This may suggest that at ratios as low as 351:1 in thisparticular formulation, excess DDAB that is not neutralized by DNA findsits way to the surface, giving the particles their positive charge.

FIG. 3 demonstrates the effect of amphipathic lipid concentration onparticle encapsulation efficiency. Nanoparticles produced via thereverse micelle nanoprecipitation method are able to encapsulated up to˜18% of the initial load of 40 bp DNA in one study (represented by thedashed line; EE=mass DNA detected after particle lysis/initial mass ofDNA). The encapsulated DNA in this case represents approximately 1.4% ofthe total initial mass of components used to formulate the nanoparticle(represented by the solid line; wt % DNA=mass DNA detected afterparticle lysis/total initial mass of polymer+lipid+DNA). Note that thisrepresents the minimum expected numbers, since all of the components maynot be represented in the same ratios in the nanoparticles as in theinitial starting materials.

FIG. 4 demonstrates the effect of amphipathic lipid concentration onweight percent nucleic acids in the nanoparticles that are formed usingthe methods of the invention. In one study, the weight % (wt %) of DNA(40 bp double-stranded) in the nanoparticles (wt %=mass DNA detectedafter particle lysis/total initial mass polymer+lipid+DNA) varies withlipid concentration. Lecithin=neutral, DDAB (dimethyldioctadecylammonium bromide)=positively charged. Interestingly, use of thepositively charged DDAB lipid results in much less DNA encapsulation.

FIG. 5 demonstrates the effect of amphipathic lipid concentration onparticle encapsulation efficiency, using the same study described forFIG. 4. The encapsulation efficiency is correspondingly larger forlecithin nanoparticles as compared to DDAB, and was as high asapproximately 30% in one case (3 mg/mL lipid in THF).

FIG. 6 demonstrates the effect of amphipathic lipid concentration ontherapeutic agent release over time. A study of the release of 40 bp DNAfrom reverse micelle nanoparticles over time shows that approximately50% of the encapsulated DNA is released within 5-10 hours, with completerelease after approximately 40 hours.

FIG. 7 demonstrates cell uptake of the polymer-encapsulated reversemicelles of the invention. Transfection of pCMV-GFP using reversemicelles encapsulated in 180 nm PLA-PEG nanoparticles. Aptamer targetednanoparticles deliver plasmid DNA effectively, while controls do not.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications,websites, and other references cited herein are hereby expresslyincorporated herein in their entireties by reference.

1. Nanoparticles comprising reverse micelles encapsulating a hydrophilicnucleic acid or protein molecule, wherein the reverse micelles areencapsulated within a hydrophobic polymeric matrix.
 2. The nanoparticlesof claim 1, wherein targeting moieties are attached to the nanoparticle.3. The nanoparticles of claim 2, wherein the targeting moiety is anaptamer.
 4. The nanoparticles of claim 1, wherein the polymeric matrixcomprises two or more polymers.
 5. The nanoparticles of claim 1, whereinthe polymeric matrix comprises a polymer selected from the groupconsisting of polyethylenes, polycarbonates, polyanhydrides,polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides,polyacetals, polyethers, polyesters, poly(orthoesters),polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes,polyacrylates, polymethacrylates, polycyanoacrylates, polyureas,polystyrenes, polyamines, and combinations thereof.
 6. The nanoparticlesof claim 1, wherein the hydrophobic polymer forming the polymeric matrixcomprises a polyalkylene glycol.
 7. The nanoparticles of claim 1,wherein the polymeric matrix comprises poly(lactide-co-glycolide)(PLGA), polylactide (PLA), polyglycolide (PGA), or a polycaprolactone.8. The nanoparticles of claim 1, wherein the polymeric matrix comprisesa copolymer of two or more polymers.
 9. The nanoparticles of claim 1,wherein the polymeric matrix comprises a lipid-terminated polyalkyleneglycol and a polyester.
 10. The nanoparticles of claim 9, wherein thepolymeric matrix comprises lipid-terminated PEG and PLGA.
 11. Thenanoparticles of claim 10, wherein the lipid is of the Formula V

and salts thereof, wherein each R is, independently, C₁₋₃₀ alkyl. 12.The nanoparticles of claim 11, wherein the lipid is 1,2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts thereof.13. The nanoparticles of claim 1, further comprising therapeutic,prophylactic or diagnostic agent encapsulated within the hydrophobicmatrix of the nanoparticle.
 14. The nanoparticles of claim 1, whereinthe therapeutic agent is an siRNA.
 15. The nanoparticles of claim 1,wherein the reverse micelle comprises an amphipathic lipid.
 16. Thenanoparticles of claim 15, wherein the amphipathic lipid is selectedfrom the group consisting of lecithin, phosphatidylcholine, lipid A,cholesterol, dolichol, shingosine, sphingomyelin, ceramide, cerebroside,sulfatide, glycosylceramide, phytosphingosine, phosphatidylethanolamine,phosphatidylglycerol, phosphatidylinositol, phosphatidylserine,cardiolipin, phophatidic acid, and lysophophatides.
 17. Thenanoparticles of claim 16, wherein the therapeutic agent is a nucleicacid, and the ratio of amphipathic lipid to nucleic acid isapproximately 33:1.
 18. The nanoparticles of claim 2, wherein thetargeting moiety binds prostate specific membrane antigen (“PSMA”). 19.The nanoparticles of claim 18, wherein the targeting moiety binds PSMAon cells selected from the group consisting of prostate cancer,non-small cell lung cancer, colorectal carcinoma, and glioblastomacells.
 20. The nanoparticles of claim 1, wherein the therapeutic agentis a nucleic acid.
 21. A pharmaceutical composition comprising thenanoparticles of claim 1 and a pharmaceutically acceptable excipient.22. A method of preparing a nanoparticle of claim 1, the methodcomprising steps of providing a hydrophilic therapeutic agent,diagnostic agent or prophylactic agent; dissolving the agent with anamphipathic lipid in a volatile, water-miscible organic solvent; formingreverse micelles, wherein the interior of the reverse micelle ishydrophilic and contains the therapeutic agent, diagnostic agent orprophylactic agent, and the exterior of the reverse micelle ishydrophobic; adding a solution of a hydrophobic polymer to the mixtureof reverse micelles; combining the resulting mixture with a hydrophilicnon-solvent that the polymer is not soluble in to form nanoparticle byrapid diffusion of the solvent into the non-solvent and evaporation ofthe solvent.
 23. The nanoparticle formed by the method of claim
 22. 24.The nanoparticles of claim 20, wherein the targeting moiety is anaptamer.
 25. The nanoparticles of claim 2, wherein the nanoparticle hasa cancer targeting moiety bound thereto.